CN109182159B

CN109182159B - N-acyl homoserine lactone quenching and sterilizing agent and application thereof in disease prevention and control

Info

Publication number: CN109182159B
Application number: CN201810843540.4A
Authority: CN
Inventors: 陈少华; 范兴辉; 叶田; 梁梓侨; 李欣; 李�昊; 江嘉敏
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2021-04-23
Anticipated expiration: 2038-07-27
Also published as: CN109182159A

Abstract

The invention discloses aNAcyl homoserine lactone quenching bacterium and application thereof in disease prevention and control. The research of the invention finds that the ochrobactrum intermedium has quenching activity on a plurality of AHLs quorum sensing signal molecules, and is separated into a high-efficiency quenching bacterial strain D-2, so that the ochrobactrum intermedium can rapidly and obviously degrade the AHLs quorum sensing signal molecules, including OHL, OOHL, OdDHL and the like, and meanwhile, pathogenic bacteria disease symptoms depending on AHLs can be obviously relieved, and the invention has great application potential in the aspect of preventing and treating the harm of AHLs mediated pathogenic bacteria. The strain has stable activity, provides a new development approach for a treatment strategy which takes biological control instead of chemical control and blocks quorum sensing as a target without causing selection pressure, has huge popularization and application potentials in the aspect of preventing and treating pathogenic bacteria hazards depending on quorum sensing signal mediated pathogenicity, and has wide application prospects in the aspect of resisting pathogenicity biocontrol bacteria taking quorum quenching as an action mode.

Description

N-acyl homoserine lactone quenching and sterilizing agent and application thereof in disease prevention and control

Technical Field

The invention belongs to the technical field of biological control. More particularly, relates to N-acyl homoserine lactone quenching bacterium and application thereof in disease prevention and control.

Background

The genus Pectinobacterium carotovorum, subforma, Pcc, is a major member of the pathogenic bacteria of soft rot of plants (Lim J, Jee S, Lee D H, et al. Biocontrol of Pectinobacterium carotovorum subsp. carotovorum using bacteriophagae PP1[ J ]. Journal of Microbiology and Biotechnology,2013,23(8): 1147-. Pcc can not only infect a variety of temperate, subtropical crops and ornamental plants, such as: crops such as Chinese cabbage, radish, tomato, potato, carrot, pepper, celery, cauliflower and the like; the ornamental plants comprise clivia miniata, lily, violet, narcissus, cactus, etc. Moreover, medicinal plants such as typhonium giganteum and silybum marianum can be infected. Pcc the bacterial soft rot caused by the disease has a wide occurrence range, is generated in a plurality of countries such as Lianpottery, Japan, Germany, Argentina, New Zealand and China and has serious harm, and has become a worldwide disease and causes the yield reduction of related crops, causing great economic loss. Aiming at bacterial soft rot, currently, chemical pesticides such as benziothiazolinone, thifluzazole, amobam, streptomycin sulfate, zhongshengmycin and the like are mainly used for preventing and treating the bacterial soft rot. However, the use of chemical pesticides in large quantities has brought about a series of serious problems of well-known environmental pollution, ecological balance destruction, pathogenic bacteria resistance and food safety, and in addition, the abuse of antibiotics also causes the generation of microbial resistance. Therefore, the search for new and effective prevention strategies is urgent.

The microorganisms themselves can produce and secrete one or more chemical substances, and as the population density of the microorganisms increases, the concentrations of the chemical substances in the environment also increase, and when the concentrations of the chemical substances reach a certain threshold, certain specific genes, especially many pathogenic genes, of the microorganisms begin to be expressed. This phenomenon is known as Quorum Sensing (QS) and these chemicals are known as Quorum sensing signal molecules or Autoinducers (AIs) (Whiteley M, Diggle S P, Greenberg E P. progress in and premium of bacterial Quorum sensing research [ J ] Nature,2017,551(7680): 313-) 320.). N-Acyl Homoserine Lactones (AHLs) are quorum sensing signals specific to gram-negative bacteria, and most of the N-Acyl homoserine lactones have the same cyclic structure of homoserine lactone and Acyl side chains, but the lengths and the saturation of the Acyl side chains are different. N- (3-oxohexanoyl) -L-homoserine lactone (OHL, 3OC6HSL), N- (3-oxooctanoyl) -L-homoserine lactone (OOHL,3OC8HSL), N- (3-oxodecanoyl) -L-homoserine lactone (OdDHL,3OC12HSL) are AHLs, and the difference is that the acyl side chain length is different.

Pcc it is possible to degrade plant cell walls by synthesizing and secreting hydrolytic enzymes such as: pectinase, polygalacturonase, protease and the like attack the defense system of the plant to destroy the tissue structure of the plant, obtain water and nutrient components to support self growth and reproduction, and after infection is successful, host plants show symptoms related to soft rot. Pcc also have quorum sensing and regulate the expression of hydrolases associated with their pathogenicity. Pcc it can synthesize and secrete an AHL-OHL, the density of which increases with Pcc population density, when the OHL reaches a certain threshold, it binds to LuxR family transcriptional activator protein, the pathogenic factor, i.e. gene coding for extracellular enzyme starts to express, the hydrolytic enzyme related to pathogenicity is synthesized, the host plant is successfully infected, and the host suffers from soft rot (Pierson L S I, Wood D W, Pierson E A. host lactic-mediated gene regulation in plant-associated bacterial [ M ]. Webster R K. Annual Review of physiology, 1998: 207. 225.).

Quorum Quenching (QQ) is a new strategy for disease control based on Quorum sensing phenomena, namely: the quorum sensing system is interfered by a mechanism of inhibiting synthesis, accumulation and monitoring of signal molecules or carrying out enzymatic degradation or modification on the signal molecules, the expression of genes related to pathogenicity of microorganisms is inhibited, and the pathogenicity of the microorganisms is weakened, so that the aim of preventing and treating diseases is fulfilled. The microbial signal molecules are degraded by Quorum sensing quenching bacteria or Quorum sensing quenching enzymes (Quorum sensing enzymes), and the Quorum sensing quenching enzyme is the most effective Quorum sensing quenching way with the lowest toxicity at present. Quorum sensing quenching is carried out by regulating a quorum sensing system to prevent and control diseases, so that selective pressure cannot be generated on microorganisms, and bacteria cannot generate drug resistance theoretically. Quorum sensing quenching is a new way for effectively preventing and treating plant bacterial diseases, and has the advantages of simple and convenient operation, economy, practicality, environmental friendliness, high efficiency, short period and the like. The development of quorum-quenching agents against different quorum-sensing signals is currently an international research hotspot.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel microbial strain with the capability of quenching quorum sensing signal molecules, namely Ochrobactrum intermedium, which has obvious and rapid degradation effect on a plurality of AHLs quorum sensing signal molecules and has huge application potential in the aspect of preventing and treating pathogen harm mediated by quorum sensing signal molecules, thereby providing a novel development approach for a control strategy which takes biological control instead of chemical control and blocks quorum sensing as a target without causing selection pressure.

The invention aims to provide application of Ochrobactrum intermedium (Ochrobactrum intermedium) in quenching microbial quorum sensing signal molecules AHLs or application in preparing products for quenching and degrading the microbial quorum sensing signal molecules AHLs; and the application in preventing and treating plant diseases which depend on the microbial quorum sensing signal molecule to mediate diseases, or the application in preparing a preventing and treating preparation of pathogenic bacteria which depend on the microbial quorum sensing signal molecule to mediate diseases.

The invention also aims to provide the ochrobactrum intermedium strain D-2 capable of quenching and degrading microbial quorum sensing signal molecules and application thereof.

The above purpose of the invention is realized by the following technical scheme:

the invention discovers for the first time that Ochrobactrum intermedium (Ochrobactrum intermedium) has the function of quenching and degrading microbial quorum sensing signal molecules, and obtains a high-efficiency degrading bacterium from a soil sample collected from a power plant in Hebei province through separation, purification, screening and identification, wherein the Ochrobactrum intermedium strain D-2 is preserved in Guangdong province microbial culture collection (GDMCC) 7/5 in 2018, and the preservation number is GDMCC No: 60409, the preservation address is No. 59 building No. 5 building of No. 100 Dazhong Tokyo, Guangzhou city.

The colony morphology of the strain D-2 is characterized in that: culturing on nutrient agar plate for 48h, the colony is white, round, convex, translucent, and has regular edge.

The morphological characteristics of the thalli observed by an electron microscope are as follows: the cells are nearly spherical and have flagella.

The physiological and biochemical characteristics of the strain D-2 are as follows: gram-negative bacteria, aerobic during growth, positive reaction in a catalase test and an oxidase test, and negative reaction in a gelatin liquefaction test.

The resistance of the strain D-2 to ampicillin reaches 400 mug. multidot.mL^-1As described above, the resistance to kanamycin reached 350. mu.g/mL^-1The resistance to gentamicin and streptomycin reaches 40 mu g/mL^-1The resistance to chloramphenicol reaches 10 mu g/mL^-1And no resistance to tetracycline.

Experimental results show that the ochrobactrum intermedium strain D-2 has quenching activity on medium-long-chain AHLs quorum sensing signal molecules and can obviously and rapidly degrade medium-long-chain AHLs. Strain D-2 was able to grow normally in basal salt medium with quorum sensing signal molecule OHL as the sole carbon source at concentrations as high as 0.4mM and completely decompose OHL within 32 h. Has great application potential in preventing and treating pathogenic bacteria harm mediated by AHLs group body induction signal molecules.

Therefore, the following applications should be within the scope of the present invention:

the application of ochrobactrum intermedium in quenching microbial quorum sensing signal molecules AHLs.

The application of ochrobactrum intermedium in preparing a product for quenching and degrading quorum sensing signal molecules AHLs.

The application of ochrobactrum intermedium in preventing and treating plant diseases which depend on microbial quorum sensing signal molecule mediated pathogenesis.

The application of ochrobactrum intermedium in preparing the preparation for preventing and treating pathogenic bacteria which depend on the microbial quorum sensing signal molecules to cause diseases.

Preferably, in any of the above applications, the ochrobactrum intermedium is the isolated ochrobactrum intermedium strain D-2 of the present invention.

In addition, the microbial quorum sensing signal molecule is N-Acyl Homoserine Lactones (AHLs).

Preferably, the microbial quorum sensing signal molecules are medium-long-chain AHLs.

More preferably, the microbial quorum sensing signal molecules are AHLs signal molecules OHHL, OOHL, and/or OdDHL.

Preferably, the pathogenic bacteria that are pathogenic depending on the microbial quorum sensing signal molecule are: a strain of Dickaurella (Dickeya sp.), a strain of Pectinobacterium (Pectiobacterium sp.), or a strain of Pseudomonas aeruginosa (Pseudomonas aeruginosa).

The plant disease includes soft rot, such as potato soft rot or carrot fleshy root soft rot. More particularly soft rot caused by pectobacterium (e.g. Z3-3).

In addition, a method for preventing and treating pathogenic bacteria diseases caused by AHLs quorum sensing signal molecules, which is to specifically perform inoculation treatment on crops by using a bacterial liquid of ochrobactrum intermedium to prevent and treat pathogenic bacteria infection and diseases caused by the AHLs quorum sensing signal molecules, and also belongs to the protection scope of the invention.

Preferably, the treatment is by inoculation treatment of the crop.

Preferably, when applied, the pH optimum of said ochrobactrum intermedium quenching AHLs is 6.5, and the temperature optimum is 30 ℃.

In addition, a series of functional microbial agents can be prepared by utilizing the ochrobactrum intermedium, such as a quenching microbial agent which can quench microbial substance sensing signal molecules and contains the ochrobactrum intermedium and/or a bacterial solution thereof, and a biocontrol agent which contains pathogenic bacteria of the ochrobactrum intermedium and/or the bacterial solution thereof and depends on the microbial substance sensing signal molecules to cause diseases, and the biocontrol agent also belongs to the protection scope of the invention. Preferably, the strain D-2 containing ochrobactrum intermedium screened by the invention.

The invention also provides a preparation method of the bacterial liquid of the strain D-2, which comprises the following steps: specifically, the strain D-2 is streaked on an LB solid culture medium flat plate, the culture is carried out for 24h at the temperature of 30 ℃, a single colony is selected and inoculated in an LB liquid culture medium for pre-culture till logarithmic phase, and the strain D-2 bacterial liquid is obtained. The concentration of the bacterial liquid is not strictly limited, and can be specifically adjusted according to the actual disease degree and the application effect.

Preferably, the LB medium is: tryptone 10.0 g.L^-15.0 g.L of yeast extract^-1Sodium chloride 10.0 g.L^-1Sterilizing at the temperature of 121 ℃ for 20min at the pH of 6.8-7.2. The LB solid medium formulation is to add 1.5% (w/v) agar to the liquid medium.

The invention has the following beneficial effects:

the research of the invention discovers that Ochrobactrum intermedium (Ochrobactrum intermedium) has quenching activity on multiple AHLs quorum sensing signal molecules, can rapidly and obviously degrade the multiple AHLs quorum sensing signal molecules, including OHL, OOHL, OdDHL and the like, has huge application potential in the aspect of preventing and treating pathogenic bacteria harm caused by AHLs mediated pathogenesis, and provides a new development approach for a treatment strategy which replaces chemical prevention by biological prevention and treats blocking quorum sensing as a target without causing selective pressure.

Meanwhile, the ochrobactrum intermedium D-2 is obtained by screening, can normally grow in a culture medium with OHL with the concentration as high as 0.4mM as a unique carbon source, can completely degrade a quorum sensing signal molecule OHL within 32h, has a remarkable biodegradation effect, can prevent and treat diseases such as bacterial soft rot and the like which are caused by AHLs, such as diseases caused by pathogenic bacteria such as the carrot soft rot Pectobacterium carotovorum subsp.

According to the invention, because the strain D-2 has the characteristic of stably and efficiently degrading quorum-sensing AHLs signal molecules in plant pathogenic bacteria, the strain D-2 can be applied to the prevention and treatment of AHLs mediated pathogenic bacteria in natural environment, thereby not only reducing the problem of pesticide abuse, but also bringing new thinking, new approaches and new methods for disease prevention and treatment.

Drawings

FIG. 1 is a colony morphology of the strain D-2 of the present invention on LB medium.

FIG. 2 is a scanning electron micrograph of the strain D-2 of the present invention.

FIG. 3 is a phylogenetic tree analysis diagram of the strain D-2 of the present invention.

FIG. 4 is a graph showing the growth of strain D-2 of the present invention in various antibiotics.

Fig. 5 is a graph showing the results of quenching activities of the strain D-2 of the present invention against different AHLs (e.coli is a negative control, B23 is a positive control), (a) is OHHL, (B) is OOHL, and (c) is OdDHL.

FIG. 6 is a graph showing growth curves and degradation curves of strain D-2 of the present invention using OHL as a sole carbon source.

FIG. 7 shows the disease onset of the strains D-2, E.coli, B23 of the present invention after being co-inoculated with Z3-3, respectively, to potato tubers for 24 hours.

Fig. 8 shows the disease of the strains D-2, E.coli, B23 of the present invention after being co-inoculated with Z3-3 for 24 h.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 acquisition and characterization of Ochrobactrum intermedium Strain D-2

1. Isolation and screening of Strain D-2

(1) Soil sample collection: soil sample collected from power plant in Hebei province as microbial source

And (3) separating the soil sample from a power plant in the province of Hebei in 2017, 8, 9 and taking the soil sample as a microbial source, bagging the soil sample, and taking the soil sample as the microbial source to bring the soil sample back to school for strain separation.

(2) Enrichment culture of the strain: a basal salt (MSM) medium was prepared, 20mL of the MSM medium was sterilized in a 250mL Erlenmeyer flask, and after cooling, a stock solution of OHL (acetonitrile as a solvent) was added under aseptic conditions so that the final concentration of OHL was 5. mu.M, and 5g of a soil sample was added at the same time, and after culturing at 30 ℃ and 200rpm for 7 days, the mixture was inoculated in 10% of an amount into a second batch of MSM medium containing 10. mu.M OHL. After culturing for 7 days under the same conditions, the cells were inoculated into MSM medium containing 20. mu.M OHL in an amount of 10%, and the culture was continued for 7 days. By analogy, the concentration of OHHL is continuously increased.

Wherein the formula of the basic salt (MSM) culture medium is as follows: 2g of ammonium sulfate; magnesium sulfate heptahydrate 0.2 g.L^-1(ii) a Calcium chloride dihydrate 0.01 g.L^-1(ii) a Ferrous sulfate heptahydrate 0.001 g.L^-1(ii) a Disodium hydrogen phosphate dodecahydrate 1.5 g.L^-1(ii) a 1.5 g.L of sodium dihydrogen phosphate^-1；pH 6.5。

(3) Strain separation and purification: the separation was performed by dilution and plate coating.

Diluting 1mL of final MSM culture medium fermentation broth with sterile water to 10% concentration^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8Then sucking 100 mul fermentation liquor with each concentration and evenly coating the fermentation liquor on an LB solid plate, culturing at 30 ℃, selecting single bacterial colonies with different bacterial colony morphologies, and repeatedly carrying out streak culture and purification on the LB solid plate until single bacterial strains are separated. The single strain is preserved at the temperature of 80 ℃ below zero, and the degradation effect on AHLs is determined by further experiments.

(4) Strain screening: strains isolated from soil samples were screened using the reporter strain CF11(Agrobacterium tumefaciens NT 1).

The strain to be screened, which is stored at minus 80 ℃, is streaked on an LB flat plate, and is cultured at 30 ℃ to activate the strain. And selecting a single colony, inoculating the single colony to a liquid LB culture medium, and culturing overnight at the temperature of 30 ℃ and the speed of 200rpm to obtain a bacterial liquid. Taking a certain volume (V is 1/OD)₆₀₀) Centrifuging the bacterial solution at 4000rpm for 10min, and removing the supernatant to obtain an OD₆₀₀The cell of value. 1 OD₆₀₀The resulting cells were inoculated into 1mL of MSM medium containing OHL as a sole carbon source, and the final concentration of OHL in the MSM medium was 20. mu.M. The reaction mixture was placed in a 2mL centrifuge tube and incubated at 30 ℃ for 24h at 200 rpm. After 24h, 5. mu.L of the reaction mixture was spotted onto the top of a 1cm wide MM agar strip, followed by a row of AHLs detectable reporter strain CF11(Agrobacterium tumefaciens NT1) in the lower row. Wherein the pH value of the MM agar strip is 6.5, and the agar strip contains 40 mu g/mL^-1X-gal of (1). The MM agar strips are placed in an incubator at 28 ℃, and the experimental results are observed after 24 hours of light-shielding culture.

Wherein the basic (MM) medium has the formula: ammonium sulfate 2 g.L^-1(ii) a Magnesium sulfate heptahydrate 0.2 g.L^-1(ii) a 0.01 g.L of anhydrous calcium chloride^-1(ii) a Ferrous sulfate 0.005 g.L^-1(ii) a Manganese chloride 0.002 g.L^-1(ii) a Dipotassium hydrogen phosphate10.5g·L^-1(ii) a Potassium dihydrogen phosphate 4.5 g.L^-1；pH 6.5。

(5) And (4) analyzing results: when the reporter strain CF11(Agrobacterium tumefaciens NT1) detects that the agar strips contain AHLs, the beta-galactosidase related gene begins to be expressed, and CF11 secretes beta-galactosamine to the environment. The beta-galactosamine can carry out enzymolysis on a colorless compound X-gal (5-bromo-4-chloro-3-indole-beta-D-galactoside) contained in the MM agar strips into galactose and a dark blue substance 5-bromo-4-indigo, and the colony of the whole reporter strain can be changed into blue by the 5-bromo-4-indigo. AHLs can diffuse on agar strips, and the diffusion distance is proportional to their concentration. In summary, the AHLs content in the original sample is analyzed according to the distance of the strain reported to turn blue from the top on the agar strip, and the shorter the distance of turning blue from the top, the better the degradation effect of the corresponding strain. And finally, screening out the strain with the best degradation effect on the AHLs according to the experimental result, and naming the strain as the strain D-2.

2. Identification and phylogenetic analysis of Strain D-2

(1) Colony morphology characteristics (as shown in figure 1): culturing on nutrient agar plate for 48h, the colony is white, round, convex, translucent, and has regular edge.

(2) Morphological characteristics of the thallus: as shown in FIG. 2, the cells were nearly spherical and had flagella.

(3) Physiological and biochemical characteristics: gram-negative bacteria, aerobic during growth, positive reaction in a catalase test and an oxidase test, and negative reaction in a gelatin liquefaction test. The physiological and biochemical identification results are shown in table 1.

TABLE 1 physiological and biochemical identification results of Strain D-2

Physiological and biochemical characteristics	Results	Physiological and biochemical characteristics	Results
				Gram stain	－	Contact enzyme	+
Anaerobic growth	－	Oxidase enzyme	+
				Liquefaction of gelatin	－

Note: -: negative reaction; +: and (4) positive reaction.

(4)16S rDNA sequence and phylogenetic analysis: the length of the 16S rDNA gene sequence of the strain D-2 is 1396bp, and the strain D-2 is compared with an NCBI database (http:// www.ncbi.nlm.nih.gov /), and the strain D-2 and Ochrobactrum intermedium have good homology (> 99%), and the phylogenetic tree is shown in figure 3.

(5) Biolog automatic analysis system identification of microorganisms:

biolog automated microbial analysis refers to the identification of the genus of a microorganism by determining its utilization of various carbon sources such as sugars, amines, vinegar, acids, alcohols, and macromolecular polymers. A96-well assay plate containing multiple dehydrated carbon sources was used for the assay. The microorganism may reduce the tetrazole redox stain to a purple color during respiration using a carbon source, resulting in a reaction pattern or "fingerprint" characteristic of the microorganism on the assay plate. The fingerprint of the microbe is collected by the instrument and then matched with the database to obtain the identification result quickly.

The result of the identification of the Biolog microorganism automatic analysis system of the strain D-2 shows that the strain D-2 is most similar to Ochrobactrum intermedium. The results of carbon source utilization by the strain D-2 are shown in Table 2.

TABLE 2 identification of carbon Source utilization by Strain D-2

Note: "-" indicates a negative reaction or not utilized; "+" indicates a positive reaction or utilization; "+/-" indicates a weak reaction.

In summary, the 16S rDNA sequence, morphological characteristics, physiological and biochemical characteristics and the Biolog microorganism automatic analysis system result of the strain D-2 are analyzed, so that the strain D-2 is identified as Ochrobactrum intermedium (Ochrobactrum intermedium) and is preserved in Guangdong province microorganism culture collection (GDMCC) 7 and 5 days 2018, and the preservation number is GDMCC No: 60409, the preservation address is No. 59 building No. 5 building of No. 100 Dazhong Tokyo, Guangzhou city.

Example 2 antibiotic susceptibility analysis of Strain D-2

In order to be able to better study the biocontrol potential of the strain D-2 obtained in example 1, we have conducted intensive studies on the biological properties of this strain. The sensitivity of strain D-2 to different antibiotics was investigated, as shown in FIG. 4.

The experimental results show that: the resistance of the strain D-2 to ampicillin reaches 400 mug. multidot.mL^-1As described above, the resistance to kanamycin reached 350. mu.g/mL^-1The resistance to gentamicin and streptomycin reaches 40 mu g/mL^-1The resistance to chloramphenicol reaches 10 mu g/mL^-1And no resistance to tetracycline. This result is beneficial for selecting suitable antibiotic in subsequent researchThe element is used as reference.

EXAMPLE 3 Strain D-2 substrate profiling

1. Strain culture and sample collection:

strain D-2 was activated on LB solid plates at 30 ℃. And selecting a single colony, inoculating the single colony to an LB liquid culture medium, and culturing overnight at the conditions of 30 ℃ and 200rpm to obtain a bacterial liquid. Taking a certain volume (V is 1/OD)₆₀₀) Centrifuging the bacterial solution at 4000rpm for 10min, and removing the supernatant to obtain an OD₆₀₀The cell of value. 1 OD₆₀₀The cells were inoculated into 1mL of MSM mineral salt medium containing different AHLs as the sole carbon source. The reaction mixtures were placed in 2mL centrifuge tubes, respectively, and the tubes were incubated at 30 ℃ for 24h at 200 rpm. After 24h, 5. mu.L of the reaction mixture was spotted onto the top of a 1cm wide MM agar strip, followed by the reporter strain CF11(Agrobacterium tumefaciens NT1) in the lower spot. And then placing the agar strips of the reaction mixture and the report strain in an incubator at 28 ℃, and observing the experimental result after culturing for 24 hours in a dark place. Wherein the agar strips have a concentration of 40 μ g/mL^-1Cutting MM plate of X-gal into strips. The content of Acyl Homoserine Lactones (AHLs) in the sample can be judged according to the distance from the top of the reported strain to turn blue on the agar strip. Thereby determining whether strain D-2 has quenching activity against different AHLs.

In the experiment, Bacillus thuringiensis subsp. israelensis B23, which is known to quench a variety of AHLs, was used as a positive control (Dong Y, Xu J, Li X, et al AiiA, an enzyme which is inactivated in the presence of the acylhomo serine amino acid quantitative-sensing signal and events of the virus of Erwinia carotovora [ J ]. Proc Natl Acad Sci USA,2000,97(7):3526 @) and Escherichia coli (Escherichia coli) which does not quench AHLs activity was used as a negative control.

2. The test results show that:

in OHHL (fig. 5a), OOHL (fig. 5b) and OdDHL (fig. 5c) test groups, the diffusion distances of the AHLs of CK and negative control e.coli were consistent, i.e. the AHLs content in the reaction mixtures were similar; the positive control B23 and the reporter strain on the agar strip of the experimental group D-2 did not turn blue, and the reaction mixture of the two did not contain AHLs, which were completely degraded by the strain D-2. The strain D-2 is shown to have quenching activity on AHLs signal molecules OHL, OOHL and OdDHL.

EXAMPLE 4 determination of the relationship between growth and degradation of OHL by Strain D-2

1. Selecting single colony of strain D-2, inoculating to LB culture medium, pre-culturing until logarithmic phase, and taking out certain volume (V is 1/OD)₆₀₀) Centrifuging the bacterial solution at 4000rpm for 10min, and removing the supernatant to obtain an OD₆₀₀The cell of value. The cells were inoculated into 20mL of MSM medium, and OHL stock solution was added thereto so that the final concentration thereof was 0.4mM, and culture was carried out at 30 ℃ and 200rpm, with a time-lapse sampling. Samples were collected at different time points and OD was determined₆₀₀The value indicates the growth of the strain D-2, and the amount of residual OHL measured by HPLC indicates the degradation of OHL by the strain D-2.

2. HPLC determination method

HPLC: waters 2695. A chromatographic column: kinetex EVO C18 reverse phase chromatography column (250. mu. m.times.4.6 mm. times.5 μm). Flow rate: 0.5 mL/min^-1. Column temperature: at 30 ℃. Mobile phase: acetonitrile: water 30: 70 (v: v). Detection wavelength: 210 nm. Sample introduction amount: 20 μ L.

3. And (4) analyzing results:

HPLC analysis showed that OHL was completely degraded at 32h, as shown in FIG. 6.

In conclusion, the degradation of 3OC6HSL was positively correlated with the growth of strain D-2, which rapidly entered the logarithmic phase of growth with no lag phase in the presence of OHHL. The strain degrades OHL fastest in 0-24 h, the strain is cultured for 32h, and the OHL is completely decomposed. In contrast, the natural degradation rate of OHL within 24h was less than 40%. The result shows that the ochrobactrum intermedium D-2 has obvious and rapid degradation effect on OHL and has great application potential in the aspect of preventing and treating the pathogenic bacteria harm mediated by OHL.

EXAMPLE 5 biocontrol Effect of Strain D-2 on Potato Soft rot disease

In this example, the biocontrol effect of strain D-2 on AHLs-dependent pathogenic bacteria was studied, taking as an example the pathogenic bacterium, Pectiobacter carotovorus subsp.

In the inoculation test, the quenching bacteria D-2, the Escherichia coli E.coli and the Bacillus thuringiensis B23 are all safe and non-pathogenic bacteria.

1. The bacillus thuringiensis B23, the escherichia coli E.coli, the strain D-2 and the carrot soft rot pectobacterium Z3-3 are streaked on an LB solid plate and cultured at 30 ℃. Single colonies on the plates were picked up, inoculated into liquid LB medium, and cultured overnight at 30 ℃ and 200rpm to obtain bacterial solutions.

The bacterial liquid of B23, E.coli, strain D-2 and pathogenic bacteria Z3-3 is adjusted to 1.0 x 10 by using liquid LB⁷cfu·mL^-1. Mixing the Z3-3 with B23, E.coli, D-2 and liquid LB culture medium respectively in a certain proportion, and respectively inoculating 5 mu L of mixed bacterial liquid onto the potatoes. Namely, four experimental groups of LB + Z3-3, Z3-3+ E. coli, Z3-3+ B23 and Z3-3+ D-2 are respectively arranged. The inoculated potatoes are placed in a biochemical incubator at 28 ℃, and after 24 hours, the disease condition is observed and photographed.

2. The results are shown in FIG. 7, the potato tuber lesion areas of the Z3-3+ LB and Z3-3+ E. coli experimental groups are obviously larger than those of the Z3-3+ B23 and Z3-3+ D2 experimental groups, and the disease is serious. Namely, the disease symptoms of the soft rot disease are obviously reduced when the quenching bacterium D-2 and the pathogenic bacteria are inoculated together compared with the disease symptoms when the pathogenic bacteria are inoculated independently. Experimental results show that the strain D-2 has a good biological control effect on potato soft rot caused by the pectobacterium Z3-3.

EXAMPLE 6 biocontrol Effect of Strain D-2 on carrot Soft rot

In this example, the biocontrol effect of strain D-2 on pathogenic bacteria causing soft rot of carrot was studied by taking as an example the pathogenic bacterium, Pectiobacter carotovorus subsp.

The bacterial liquid of B23, E.coli, strain D-2 and pathogenic bacteria Z3-3 is adjusted to 1.0 x 10 by using liquid LB⁷cfu·mL^-1. Respectively mixing the Z3-3 with B23, E.coli, D-2 and liquid LB culture medium according to a certain proportion, and respectively inoculating 5 mu L of mixed bacteria liquid to the carrot fleshy root slices. Namely, four experimental groups of LB + Z3-3, Z3-3+ E. coli, Z3-3+ B23 and Z3-3+ D-2 are respectively arranged. The inoculated carrot slices are placed in a biochemical incubator at 28 ℃, and after 24 hours, the disease condition is observed and photographed.

2. The result is shown in figure 8, plant tissues of the carrot fleshy roots of the Z3-3+ LB and Z3-3+ E. coli experimental groups are rotten, while the carrot fleshy roots of the Z3-3+ B23 and Z3-3+ D2 experimental groups only have water stain-shaped disease spots and do not have the symptom of rotting of the plant tissues, namely the symptom of the carrot soft rot disease is obviously relieved when the quenching bacterium D-2 and the pathogen Z3-3 are inoculated together compared with the symptom when the pathogen Z3-3 is inoculated independently. Experimental results show that the quenching bacteria D-2 has a good biological control effect on carrot fleshy root soft rot caused by the pectobacterium Z3-3.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. An Ochrobactrum intermedium (Ochrobactrum intermedium) strain D-2 capable of quenching and degrading microbial quorum sensing signal molecules, which is preserved in Guangdong province microbial culture Collection (GDMCC) 7-5 days in 2018 and has the preservation number of GDMCC No: 60409, the preservation address is No. 59 building No. 5 building of No. 100 Dazhong Tokyo, Guangzhou city.

2. Use of the ochrobactrum intermedium strain D-2 according to claim 1 for quenching quorum sensing signal molecules or for preparing a product for quenching quorum sensing signal molecules;

the microbial quorum sensing signal molecule is N- (3-oxohexanoyl) -L-homoserine lactone, N- (3-oxoctanoyl) -L-homoserine lactone and/or N- (3-oxocanoyl) -Lhomoserine lactone.

3. Use of the ochrobactrum intermedium strain D-2 according to claim 1 for controlling plant diseases that are pathogenic mediated by a quorum sensing signal molecule, or for preparing a control agent for pathogenic bacteria that are pathogenic by a quorum sensing signal molecule;

4. A method for controlling pathogenic bacterial diseases which depend on pathogenic microorganism quorum sensing signal molecules, which is characterized in that the bacterial liquid of the ochrobactrum intermedium strain D-2 of claim 1 is used for carrying out inoculation treatment on crops;

5. A quencher for quenching an quorum sensing signal molecule, which comprises the ochrobactrum intermedium strain D-2 and/or a bacterial solution thereof according to claim 1;

6. A biocontrol agent for pathogenic bacteria which cause diseases by means of quorum sensing signal molecules, comprising the Ochrobactrum intermedius strain D-2 and/or a bacterial solution thereof according to claim 1;