CN108823130A - Inhibit plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI and application - Google Patents
Inhibit plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI and application Download PDFInfo
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Abstract
The present invention, which provides, inhibits plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI and application, and biocontrol bacterial strain classification naming is bacillus subtilis (Bacillus subtilis) GacAI, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), preservation day：On June 11st, 2018, deposit number：CGMCC No.15939, GacS/GacA system expression in plant pathogenetic bacteria can be effectively suppressed in the bacillus subtilis GacAI secondary metabolite, to influence the expression of the encoding genes such as Disease-causing gene such as transelminase, soft rot of Chinese cabbage can be effectively prevented by the suspending agent that the bacillus subtilis GacAI is effective component preparation, 84.1% is reached to the control efficiency of soft rot of Chinese cabbage.
Description
Technical field：
The present invention relates to field of biotechnology, in particular to a kind of inhibition bacterium GacS/GacA two-component regulator control system is raw
Anti- bacterial strain GacAI and its application.
Technical background:
Bacterium survival ability in specific habitat needs to coordinate the expression of appropriate gene with the variation of response environment.To answer
To complicated external environment, bacterium has evolved a variety of induction regulator control system such as GacS/GacA two-component regulatory system (two-
component regulatory system；TCS), monitoring external environment variation, and adaptation appropriate is made to environmental change
Property reaction.GacS/GacA system is by induction kinases (sensor kinase) GacS and the corresponding response regulatory factor
(response regulator) GacA composition.By interacting with signaling molecule, kinases GacS autophosphorylation is incuded.Phosphorus
Phosphate group is transferred to corresponding response regulatory factor GacA, the controllable downstream targets gene of the GacA of activation by the GacS of acidification
Expression (Heeb, S., and D.Haas. (2001) .Regulatory roles of the GacS/GacA two-
component system in plant-associated and other Gram-negative
bacteria.Mol.Plant-Microbe Interact.14:1351-1363.).The regulator control system is by researcher at present
One of Pathogenicity of Bacteria research hotspot of attention.
The expression of various plants gram negative pathogenic Pathogenicity of Bacteria related gene is by the stringent of GacS/GacA system
Regulation.Such as：Cause the carrot soft rot Pectinatus of the various plants soft rot such as potato, wild cabbage, carrot
(Pectobacterium carotovorum subsp.carotovorum, Pcc) synthesizes a large amount of when infecting host plant
Hydrolase, such as：Pectase, polygalacturonase and pectate lyase etc. destroy plant cell wall, cause soft rotten disease
Shape.These destroy the ectoenzyme of host cell wall and other generations with the virulence factor of host plant interaction by GacS/
Stringent regulation (Cui, Y., A.Chatterjee, and A.K.Chatterjee. (2001) .Effects of of GacA system
the two-component system comprising GacAand GacS of Erwinia carotovora
subsp.carotovora on the production of global regulatory rsmB RNA,
extracellular enzymes,and HarpinEcc.Mol.Plant-Microbe Interact.14:516-526.)。
In addition to soft rot Pectinatus, GacS/GacA system also influences pathogenic expression in other plant pathogenetic bacteria.Such as：Cause
The xylella fastidiosa (Xylella fastidiosa) of grape Pearls, with pathogenic correlation factor such as adhesive ability and life
Formation of object film etc. by GacS/GacA system regulation, the pathogenicity of GacS/GacA system sudden change body significantly reduce (Shi,
X.Y.,C.K.Dumenyo,R.Hernandez-Martinez,et al.(2009).Characterization of
regulatory pathways in Xylella fastidiosa:genes and phenotypes controlled by
gacA.Appl.Environ.Microbiol.75:2275-2283.).Pseudomonas syringae (Pseudomonas syringae)
The generation of colonizing on host plant and a variety of virulence factors be also required to GacS/GacA system participation (Chatterjee,
A.,Y.Cui,H.Yang,et al.(2003).GacA,the response regulator of a two-component
system,acts as a master regulator in Pseudomonas syringae pv.tomato DC3000by
controlling regulatory RNA,transcriptional activators,and alternate sigma
factors.Mol.Plant-Microbe Interact.16:1106-1117.).Plant pathogenetic bacteria Dickeya
GacS/GacA system passes through the table for influencing pectin lyase, protease, cellulase and three type excretory systems in dadantii
It reaches, play an important role (Yang, S.Q., Q.Peng, Q., Zhang, et al. (2008) in the bacterium pathogenic course
.Dynamic regulation of GacAin type III secretion,pectinase gene expression,
pellicle formation,and pathogenicity of Dickya dadantii(Erwinia chrysanthemi
3937).Mol.Plant-Microbe Interact.21:133-142.).Equally, rice leaf spot bacteria (Xanthomonas
Oryzae pv.oryzae) in missing GacS/GacA two-component regulator control system can significantly affect the motility of bacterial strain, and bacterium is transported
Dynamic property and the bacteria pathogenic closely related (Xu Jingsheng, Wu Maosen, what morning sun (2010) rice leaf spot bacteria two-component regulatory
Systems response regulator gene gacAxooFunction Identification Plant Pathology .40 (3):282-289.).Above-mentioned report shows
The pathogenic regulation of GacS/GacA system wide participation pathogenetic bacteria.
The antibiotic such as certain environmental factors, tricarboxylic acid cycle mesostate and azithromycin can influence GacS/GacA
Two-component regulator control system.If low ph value is less than under 5.5 environmental conditions, GacS/GacA system in bacterial strain Escherichia coli
Congener --- BarA/UvrY system is in close state；Azithromycin (azithromycin) be widely used in it is clinical big
Cyclic lactone class antibiotic.Although azithromycin does not influence human body opportunistic pathogen under clinical use concentration conditions
The growth of P.aeruginosa, but azithromycin mainly pass through inhibit gacA gene translation and small RNA molecular RsmY and
Transcription (Perez-Martinez, I.and D.Haas. (2011) .Azithromycin inhibits of RsmZ
expression of the GacA-dependent small RNAs RsmY and RsmZ in Pseudomonas
aeruginosa.Antimicrob.Agents Chemother.55:3399-3405.), and then can treat by
Infection caused by P.aeruginosa.
The prevention and treatment of crop bacterium disease at present is mainly to use based on conventional antibiotic.The conventional antibiotic mechanism of action is general
It is direct kill pathogenetic bacteria, a large amount of use also leads to the environmental securities such as pathogen drug resistance raising.Bis- groups of GacS/GacA
Part regulator control system plays a key effect in various plants pathogenetic bacteria pathogenic course, and the missing of the system has no effect on cause of disease
The growth of bacterium.Therefore screening inhibits the secondary metabolite resource of GacS/GacA double factor regulator control system, dedicated in prevention and treatment
Bacterial disease is stated to have broad application prospects.
Summary of the invention:
The purpose of the present invention is to provide a kind of bacillus subtilis GacAI and its answering in terms of preventing and treating bacterial disease
With.
Above-mentioned purpose is achieved by the following technical solution：
A kind of bacillus subtilis, classification naming are bacillus subtilis (Bacillus subtilis) GacAI, preservation
Unit：China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), preservation day：June 11 in 2018
Day, deposit number：CGMCC No.15939.
The secondary metabolite of bacillus subtilis GacAI CGMCC No.15939 secretion can inhibit in pathogenetic bacteria
The expression of GacS/GacA system, to influence Pathogenic.
Bacillus subtilis GacAI fermentation liquid is prepared into the prevention and treatment that biocontrol agent carries out soft rot of Chinese cabbage.
The biocontrol agent is suspending agent, by bacillus subtilis GacAI fermentation liquid, N- dodecyl-beta-amino third
Sour sodium, hydroxymethyl cellulose, acid hydrolyzed casein, sulfuric acid benzene sulfonic acid sodium salt and peptide glycan composition.
Above-mentioned suspending agent consists of the following compositions：
Bacillus subtilis GacAI fermentation liquid 1000mL
N- dodecyl-Beta-alanine sodium 20g
Hydroxymethyl cellulose 3g
Acid hydrolyzed casein 1g
Sulfuric acid benzene sulfonic acid sodium salt 2g
Peptide glycan 0.3g
Spore content is not less than 55 × 10 in bacillus subtilis GacAI fermentation liquid8 CFU/mL。
Each component partial size is not more than 5 μm in suspending agent.
Beneficial effects of the present invention are：Plant can be effectively suppressed in bacillus subtilis GacAI secondary metabolite in the present invention
GacS/GacA system expression in object pathogenetic bacteria, to influence the expression of the encoding genes such as Disease-causing gene such as transelminase.
Soft rot of Chinese cabbage can be effectively prevented by the suspending agent that bacillus subtilis GacAI is effective component preparation, to the soft corruption of Chinese cabbage
The control efficiency of disease reaches 84.1%.
Detailed description of the invention：
Fig. 1 show the expression that bacillus subtilis GacAI secondary metabolite inhibits tiny RNA encoding gene rsmB；Fig. 2
Showing bacillus subtilis GacAI secondary metabolite inhibits Chinese cabbage soft rot bacteria intervention school-based signaling molecule to produce
It is raw；Fig. 3 show bacillus subtilis GacAI secondary metabolite and inhibits Chinese cabbage soft rot bacteria Pcc Z3-3 pathogenic.
Specific embodiment：
The present invention is illustrated to be apparent, below with specific example in detail technical solution of the present invention, but the present invention is simultaneously
It is not limited to this.
Embodiment 1
Bacillus subtilis GacAI secondary metabolite inhibits phytopathogen Pectobacterium carotovorum
The expression of tiny RNA encoding gene rsmB in subsp.carotovorum (Pcc) Z3-3.
GacS/GacA system is just regulating and controlling the expression of tiny RNA encoding gene rsmB.By rsmB gene promoter region through PCR
It after amplification, is cloned on promoter function analysis plasmid pRG970Gm, to obtain the transcribed reporter structure (rsmB- of rsmB
LacZ), the expression of this report plasmid is by GacS/GacA system.By bacillus subtilis GacAI after 28 DEG C are cultivated 1 week, supernatant
It is extracted with isometric ethyl acetate, is resuspended in the dimethyl sulfoxide (DMSO) of 0.1mL after extract rotary evaporation.It takes
5 μ L extracts are added in 2mL Pcc Z3-3 (p970Gm-rsmBp) and its gacA gene mutation body (p970Gm-rsmBp)
(OD600=0.1), after cultivating 12h, betagalactosidase activity in examining report bacterium.
Referring to Fig. 1, bacillus subtilis GacAI extracting solution can significantly inhibit tiny RNA encoding gene in pathogen Z3-3
The expression of rsmB, inhibiting rate is up to 80%.And GacAI extract is added in Z3-3gacA mutant does not influence the table of rsmB gene
It reaches, shows that GacAI extract passes through the expression of GacS/GacA systematic influence rsmB gene.
Embodiment 2
Bacillus subtilis GacAI secondary metabolite inhibits colony induction signaling point in phytopathogen Pcc Z3-3
Son.
After bacterial strain Z3-3 is incubated overnight in LB culture solution, by 1：1000 dilutions are inoculated into containing various concentration GacAI times
In the LB culture solution of raw metabolin.After 28 DEG C of culture 12h, the isometric ethyl acetate of supernatant is extracted, extract rotation is steamed
It is redissolved in 100 μ L methanol after dry.3 μ L samples are taken to drip to quorum sensing report bacterium Agrobacterium
On the detection plate of tumefaciens NTL4 (pZLR4) and X-gal, after being incubated overnight, experimental result is recorded.
Referring to fig. 2, Z3-3 quorum sensing is detected using population system reporting bacterial strain A.tumefaciens NTL4 (pZLR4)
System signal molecule finds that the GacAI extract that 10 μ L concentration are 30 μ g/mL is added in detection plate can significantly reduce Z3-3 groups
Body-sensing answers system signal molecule to generate；And then can almost it inhibit when adding the GacAI extract that 40 μ L concentration are 30 μ g/mL
Bacterial strain Z3-3 intervention school-based signaling molecule generates.
Embodiment 3
Bacillus subtilis GacAI secondary metabolite inhibits plant pathogenetic bacteria Pcc Z3-3 pathogenic.
It is pressed after being incubated overnight bacterial strain Z3-3 for the influence pathogenic to Pcc Z3-3 of detection GacAI secondary metabolite
1：1000 ratios are transferred in fresh LB culture solution, 28 DEG C of cultures to OD600=1.0.Take 5 μ L bacterium solutions and various concentration
GacAI secondary metabolites mix after, be inoculated on Chinese cabbage, 28 DEG C of moisturizing cultures for 24 hours after, measure Lesion size.
Referring to Fig. 3, compared with the control, wild mushroom can clearly result in cabbage leaves and rot；It is 30 μ g/mL by 10 μ L concentration
GacAI extract and wild-type strain Z3-3 combined inoculation Chinese cabbage after, it is pathogenic to can obviously reduce Z3-3, and 40 μ L are dense
After degree is the GacAI extract of 30 μ g/mL and wild-type strain Z3-3 combined inoculation Chinese cabbage, bacterial strain Z3-3 cause can be completely inhibited
Characteristic of disease.These are the result shows that the secondary metabolites that bacterial strain GacAI is generated can influence the pathogenic of bacterial strain Z3-3.
Embodiment 4
The preparation of suspension
Bacillus subtilis GacAI fermentation liquid is prepared, spore content is not less than 55 × 10 when fermentation ends8CFU/mL is pressed
Following ratios prepare suspending agent.
Bacillus subtilis GacAI fermentation liquid 1000mL
N- dodecyl-Beta-alanine sodium 20g
Hydroxymethyl cellulose 3g
Acid hydrolyzed casein 1g
Sulfuric acid benzene sulfonic acid sodium salt 2g
Peptide glycan 0.3g
Effective component is the secondary metabolite that bacillus subtilis GacAI is generated in the suspending agent, and the generation of gemma can
The effect of extending preparation.N- dodecyl-Beta-alanine sodium is auxiliary agent and dispersing agent in suspending agent；Hydroxymethyl cellulose is to increase
Thick dose, be the protective agent of effective component；The partial size of each component is not more than 5 μm in suspending agent.
Embodiment 5
Control in field effect test of the suspending agent to soft rot of Chinese cabbage
Test site：Nanning Guangxi University Experimental Base.Breeds of Chinese cabbage：Green miscellaneous No. three.Test sets 3 processing,
Processing 1 is GacAI suspending agent, 100 times of dilutions；Processing 2 is 20% Thiodiazole-copper, 500 times of dilutions；Processing 3 compares for clear water,
Each processing sets 3 repetitions.Processing mode：Dilution is spraying.Administration time：On December 10th, 2017, on December 30th, 2017,
On January 20th, 2018, interval time are 20 days.It searches into a matter：On March 25th, 2018.
Test result shows that, (referring to table 1), suspending agent of the present invention has the soft rot of Chinese cabbage of Guangxi University proving ground
Preferable control efficiency, there are significant difference (p < 0.01) between comparison medicament, to soft rot of Chinese cabbage preventive effect up to 84.1%.
Table 1 is correlation data of the suspending agent of the present invention to the control in field effect of Guangxi University's base soft rot of Chinese cabbage.
| Processing | Disease incidence % | Preventive effect % |
| Suspending agent of the present invention | 13.3A | 84.1 |
| 20% Thiodiazole-copper | 20.9B | 74.9 |
| Clear water | 83.4C | - |
Claims (6)
1. inhibiting plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI and application, which is characterized in that
The biocontrol bacterial strain classification naming is bacillus subtilis (Bacillus subtilis) GacAI, depositary institution：The micro- life of China
Object culture presevation administration committee's common micro-organisms center (abbreviation CGMCC), preservation day：On June 11st, 2018, deposit number：
CGMCC No.15939。
2. inhibition plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI according to claim 1 and
Using, which is characterized in that the secondary metabolite of bacillus subtilis GacAI CGMCC No.15939 secretion can inhibit cause of disease
The expression of GacS/GacA system in bacterium, to influence Pathogenic.
3. inhibition plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI according to claim 1 and
Using, which is characterized in that bacillus subtilis GacAI fermentation liquid is prepared into biocontrol agent and carries out the anti-of soft rot of Chinese cabbage
It controls.
4. inhibition plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI according to claim 3 and
Using, which is characterized in that the biocontrol agent is suspending agent, by bacillus subtilis GacAI fermentation liquid, N- dodecyl-
Beta-alanine sodium, hydroxymethyl cellulose, acid hydrolyzed casein, sulfuric acid benzene sulfonic acid sodium salt and peptide glycan press following component proportion group
At：
5. inhibition plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI according to claim 3 and
Using, which is characterized in that spore content is not less than 55 × 10 in bacillus subtilis GacAI fermentation liquid8CFU/mL。
6. inhibition plant pathogenetic bacteria GacS/GacA two-component regulator control system biocontrol bacterial strain GacAI as described in claim 5 and
Using, which is characterized in that each component partial size is not more than 5 μm in suspending agent.
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2018
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