CN107893040B

CN107893040B - Microbial quorum sensing signal molecule degrading bacterium and application thereof in disease control

Info

Publication number: CN107893040B
Application number: CN201711248767.6A
Authority: CN
Inventors: 陈少华; 范兴辉; 张炼辉; 王惠杉; 叶田; 阳芳; 李绮婷
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2017-11-30
Filing date: 2017-11-30
Publication date: 2020-08-07
Anticipated expiration: 2037-11-30
Also published as: CN107893040A

Abstract

The invention discloses a microbial quorum sensing signal molecule degrading bacterium and application thereof in disease control. The pseudomonas nitroreducens are pseudomonas nitroreducens (a)Pseudomonas nitroreducens) The strain W-7 is preserved in a China center for type culture collection (CCTCC NO: M2017649) in 11/2/2017, has stable activity, can grow by taking quorum sensing signal molecules AH L s such as OdDH L and the like as a unique carbon source, has obvious and rapid degradation effect on the signal molecules, has a good degradation effect on other quorum sensing signal molecules such as OHH L and DSF, has the characteristics of simple culture method, high growth speed, difficult variation and strong adaptability, and has huge popularization and application potentials in the aspects of quorum quenching direction, soft rot disease inhibition and pathogenic bacteria hazard control depending on quorum sensing signal mediated pathogeny.

Description

Microbial quorum sensing signal molecule degrading bacterium and application thereof in disease control

Technical Field

The invention belongs to the technical field of biological control. More particularly, relates to a microbial quorum sensing signal molecule degrading bacterium and application thereof in disease control.

Background

Effective control of phytopathogens has been an important problem in agricultural production, for example, Erwinia sp and dicamba sp bacteria, which are gram-negative phytopathogens. Among them, the genus dicamba is a new genus of plant pathogenic bacteria established in recent years, and its model bacteria is the original erwinia chrysanthemi (Erwiniachrysanthemi). The genus Dicka (Dickeya sp.) can cause serious diseases of various important food crops and economic crops, such as bacterial basal rot of rice, bacterial soft rot of banana, bacterial soft rot of potato and the like, and cause yield reduction of related crops, thereby causing serious economic loss. Soft rot is an important agricultural disease, and no effective control measure is available in the production at present.

At present, the prevention and treatment measures for the soft rot pathogenic bacteria at home and abroad are mainly chemical prevention and treatment, such as: the chemical pesticides such as benziothiazolinone, thifluzazole, amobam, streptomycin sulfate and zhongshengmycin are widely used, but a series of well-known serious problems such as environmental pollution, ecological balance destruction, pathogenic bacteria drug resistance and food safety are brought about by the mass use of the chemical pesticides, and in addition, the generation of microbial drug resistance is caused by the abuse of antibiotics. Therefore, the search for new and effective prevention strategies is urgent.

Quorum Sensing (QS) refers to a phenomenon that microorganisms exchange information among themselves through signal molecules (self-inducing agents), QS is widely present in microbial populations and can regulate the expression of specific genes, especially a plurality of pathogenic genes, such as the plant pathogenic bacteria Dickeya zeae EC1 is Quorum Sensing and related to pathogenicity, the signal molecules are Acyl homoserine lactones (N-Acyl homoserines, AH L s) (Hussain MB, Zhang H B, Xu J L, et al, the Acyl-homoserines lactone genes lactic-type genes modification and viral gene clone bacteria cell and bacterium [ J ] Bac 2008, 190 (3): 1045) and the strain Escherichia coli, Pseudomonas solanacearu, Escherichia coli, Pseudomonas aeruginosa, Escherichia coli, etc. (3)' 567) are commonly present in a loop Sensing signal of Escherichia coli, Pseudomonas aeruginosa, Escherichia coli, and the like, the presence of a loop Sensing signal of a loop inducing, such as a loop inducing, wherein the L2, the presence of a signal of a loop inducing, the L2, Escherichia coli, the L2, the L-expressing a loop inducing, the L2, the L-Acyl, E, E.E, E.

Quorum sensing Quenching (QQ) prevents the effective accumulation of signal molecules by Quenching the signal molecules of pathogenic bacteria, and fails to activate the expression of pathogenic genes of pathogenic bacteria when the concentration of the signal molecules is below a certain threshold, thereby disrupting intercellular communication and disrupting the Quorum sensing system. Researches show that the disease is controlled by a quorum sensing quenching method, and no selective pressure is generated on pathogenic bacteria, so that the pathogenic bacteria are not easy to generate drug resistance. A biological control strategy (called as colony quenching) aiming at a microbial flora response system is a new way for effectively controlling plant bacterial diseases, and has the advantages of simple and convenient operation, economy, practicability, 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 overcome the defects and technical shortcomings of the prior art, and provide a nitroreduction Pseudomonas (Pseudomonas nitroreducens) with the capacity of efficiently degrading quorum sensing signal molecules, which has obvious and rapid degradation effect on quorum sensing signal molecules such as AH L s and DSF, and has huge application potential in preventing and treating pathogen harm mediated by quorum sensing signal molecules, so that a new development approach is provided for a prevention and treatment strategy which replaces chemical prevention and treatment with biological prevention and treatment and takes blocking quorum sensing as a target without causing selection pressure.

The invention aims to provide a Pseudomonas nitroreducens (Pseudomonas nitroreducens) strain W-7 capable of degrading microbial quorum sensing signal molecules.

The invention also aims to provide application of pseudomonas nitroreducens in degrading microbial quorum sensing signal molecules.

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

a nitroreduction pseudomonas (Pseudomonas nitroreducens) strain W-7 capable of degrading microbial quorum sensing signal molecules is preserved in China center for type culture collection (CCTCC NO) in 11-2 months in 2017, and the preservation number is CCTCC NO: m2017649.

The strain is obtained by manually screening, separating and purifying sewer sewage samples collected from Guangzhou city, and the strain W-7 is identified to be Pseudomonas nitroreducens (Pseudomonas nitroreducens) through morphological characteristics, physiological and biochemical characteristics, 16S rDNA phylogenetic analysis and a Biolog microorganism automatic analysis system of the strain.

The colony morphology of the strain W-7 is characterized in that: culturing for 48h on a nutrient agar plate, wherein the bacterial colony is light yellow, flat, smooth and translucent in surface and neat in edge: the cells were cultured in a nutrient broth for 48 hours to form a diffuse turbidity.

The morphological characteristics of the thalli observed by an electron microscope are as follows: the cells were rod-shaped and had 1 polar flagellum.

The physiological and biochemical characteristics of the strain W-7 are as follows: gram-negative bacteria, aerobic bacteria, catalase test, oxidase test, fluorescent pigment test, nitrate reduction test, nitrate gas production test, hemolytic test, gelatin liquefaction test, hydrogen sulfide test and starch hydrolysis test; the optimum growth temperature is 30 ℃, and the optimum pH is 7.0.

The resistance of the strain W-7 to ampicillin reaches more than 400 mu g/m L, the resistance to gentamicin, streptomycin and chloramphenicol reaches more than 200 mu g/m L, and the resistance to tetracycline reaches 10 mu g/m L.

Experimental research shows that the pseudomonas nitroreducens strain W-7 has obvious and rapid degradation effect on quorum sensing signal molecules OdDH L, can grow normally in a culture medium with OdDH L with the concentration as high as 0.2mM as a unique carbon source, and can completely decompose quorum sensing signal molecules OdDH L with the initial concentration of 0.2mM within 48 hours.

Therefore, the application of the pseudomonas nitroreducens in degrading AH L s and/or DSF signals of quorum sensing signal molecules or preparing products for degrading AH L s and/or DSF signals also belongs to the protection scope of the invention.

The application of the pseudomonas nitroreducens in preventing and treating the plant diseases caused by mediation of quorum sensing signal molecules such as AH L s or DSF and the like or the application in preparing the prevention and treatment preparation of pathogenic bacteria caused by dependence on quorum sensing signal molecules such as AH L s or DSF and the like also belongs to the protection scope of the invention.

Preferably, in any one of the above applications, the Pseudomonas nitroreducens is Pseudomonas nitroreducens strain W-7.

A method for preventing and treating the pathogenic bacteria and diseases caused by the quorum sensing signal molecules such as AH L s or DSF features that the suspension of the nitropseudomonas reducta strain W-7 is used to treat the crops to prevent the infection of pathogenic bacteria caused by AH L s.

Preferably, the treatment is by spraying or inoculating the crop.

Preferably, when the method is applied, the optimal pH value of degrading AH L s by using the pseudomonas nitroreducens is 6.5-7.2, and the optimal temperature is 28-30 ℃.

Experiments show that the nitroreduction Pseudomonas strain W-7 has a remarkable biocontrol effect on diseases caused by pathogenic bacteria depending on AH L s, such as Erwinia (Erwinia), Dickaya (Dickeya) or Pseudomonas aeruginosa, and on diseases caused by pathogenic bacteria depending on DSF, such as Xanthomonas (Xanthomonas) or Burkholderia (Burkholderia).

A degrading bacterial agent containing the degradable quorum sensing signal molecules AH L s or DSF of the pseudomonas nitroreducens strain W-7 and/or the bacterial suspension thereof and a biocontrol agent containing pathogenic bacteria of the strain W-7 and/or the bacterial suspension thereof which are dependent on AH L s or DSF to cause diseases also belong to the protection scope of the invention.

The invention also provides a preparation method of the bacterial strain W-7 bacterial suspension in the nutrient-rich medium, which comprises the steps of specifically marking the bacterial strain W-7 on a L B solid medium flat plate, culturing for 12-36 h at 28-30 ℃, selecting a single bacterial colony, inoculating the single bacterial colony in a L B liquid medium for pre-culturing to a logarithmic phase, washing the obtained bacterial strain with 0.9% of sterile physiological saline and re-suspending the bacterial strain to be used as a seed suspension, inoculating the seed suspension to a L B liquid medium according to the inoculation amount of 0.5-5% (preferably 1%) of volume ratio, culturing to the logarithmic phase, re-suspending the bacterial strain with PBS buffer solution to obtain the bacterial suspension of the bacterial strain W-7, wherein the concentration of the bacterial suspension is not strictly limited, and can be specifically adjusted according to the actual disease degree and application effect.

Preferably, the L B culture medium is tryptone 10.0 g/L, yeast extract 5.0 g/L, sodium chloride 10.0 g/L, pH 6.8-7.2, and the solid culture medium formula is that 1.5% (w/v) agar is added into a liquid culture medium and sterilized at 121 ℃ for 15-25 min, L B.

The invention has the following beneficial effects:

the research of the invention discovers that the pseudomonas nitroreducens can efficiently degrade quorum sensing signal molecules AH L s and DSF, the degradation effect is obvious and quick, and the pseudomonas nitroreducens has huge application potential in preventing and treating pathogenic bacteria hazards caused by mediating AH L s and DSF, so that a new development way is provided 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 nitroreduction Pseudomonas (Pseudomonas nitroreducens) W-7 is obtained by screening, can grow normally in a culture medium with OdDH L with the concentration as high as 0.2mM as a unique carbon source, can effectively degrade quorum sensing signal molecules OdDH L in a short time, can completely decompose quorum sensing signal molecules OdDH L with the initial concentration of 0.2mM within 48 hours, and has a remarkable biodegradation effect.

According to the invention, the strain W-7 has the characteristic of stably and efficiently degrading quorum-sensing AH L s or DSF signal molecules in plant pathogenic bacteria, so that the strain W-7 can be applied to prevention and treatment of AH L s or DSF mediated pathogenic plant pathogenic bacteria in natural environment, the abuse problem of pesticides can be reduced, and a new thought, a new path and a new method are provided for prevention and treatment of plant diseases.

Drawings

FIG. 1 is a colony morphology of the strain W-7 of the present invention on L B medium.

FIG. 2 is a colony morphology of the strain W-7 of the present invention on a blood plating medium.

FIG. 3 is a scanning electron micrograph of the strain W-7 of the present invention.

FIG. 4 is a phylogenetic tree analysis diagram of the strain W-7 of the present invention.

FIG. 5 is a graph showing the growth of the strain W-7 of the present invention in various antibiotics.

FIG. 6 is a graph showing the measurement of the degrading activity of the strain W-7 of the present invention (DH5 α is a negative control, B23 is a positive control).

FIG. 7 shows HP L C of the strain W-7 of the invention degrading OdDH L (FIG. A is a control chart of the non-inoculated strain W-7, and FIG. B, C, D, E is a high performance liquid chromatogram of the strain W-7 degrading OdDH L12 h, 24h, 36h and 48h, respectively).

FIG. 8 is a graph showing the growth curve and degradation curve of strain W-7 of the present invention using OdDH L as a sole carbon source.

FIG. 9 is a transparent hydrolysis circle diagram of strain W-7 of the present invention produced by degrading DSF on a solid MM inorganic salt medium plate with DSF as the sole carbon source.

FIG. 10 shows the disease onset of the strains W-7, DH5 α, B23 and EC1 of the present invention after being inoculated individually and together with EC1 for 30 h.

FIG. 11 shows the onset of the bacterial strains W-7 and 3937 of the present invention after they were inoculated separately and together to cabbage stalks for 24 hours.

Detailed Description

The present invention will be further described with reference to the following specific examples and the accompanying drawings, 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 present invention are commercially available.

EXAMPLE 1 acquisition and characterization of Pseudomonas nitroreducens Strain W-7

1. Isolation and screening of Strain W-7

(1) Soil sample collection: sewer sewage sample collected from Guangzhou as microbial source

The soil sample was isolated from a sewer near southern agricultural university in Guangzhou, Guangdong in 2014, 10 and 9 days, and sampled, bagged, stored as a microbial source and brought back to school for strain isolation.

(2) Enrichment culture of strain, preparing MSM culture medium, filling 50M L basal medium into 250M L triangular flask for sterilization, cooling, adding AH L s mother liquor (methanol as solvent) under aseptic condition to make final concentration of AH L s be 5 μ M, simultaneously adding soil sample 5g, shake-culturing at 30 deg.C and 200rpm for 7d, transferring 10% inoculum size into the second batch of 10 μ M AH L s MSM culture medium, transferring 10% inoculum size into 20 μ M AH L s MSM culture medium after culturing under the same condition for 7d, continuing culturing for 7d, and so on, increasing concentration of AH L s.

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

Taking 1m L final MSM culture medium fermentation liquor, and sequentially diluting the concentration of the fermentation liquor to 10 by gradient with sterile water^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8The fermentation liquor is then sucked and diluted by 100 mu L, the fermentation liquor with each concentration gradient is evenly coated on a L B solid plate, the culture is carried out at 30 ℃, single bacterial colonies with different bacterial colony morphologies are picked out, the streaking culture and the purification are carried out repeatedly on a L B solid plate until the single bacterial strain is separated, the single bacterial strain is stored at the temperature of minus 80 ℃, and the degradation effect of AH L s is further tested by experiments.

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

Streaking the strain to be screened frozen at-80 deg.C on L B solid culture medium plate, culturing overnight at 30 deg.C, selecting single colony, culturing overnight with L B liquid culture medium to obtain bacterial liquid, and collecting 1 OD₆₀₀The resulting cells were inoculated into 1m L MSM mineral salt medium containing AH L s as the sole carbon source at a final concentration of 20. mu.M AH L s, the reaction mixture was placed in a 2m L centrifuge tube, the tube was incubated at 30 ℃ and 200rpm for 24 h.24h, and then 5. mu. L of the reaction mixture was spotted onto a 200. mu. L Agrobacterium tumefaciens NT 1-coated bacterial suspension (OD)₆₀₀0.4) containing X-gal at a concentration of 40 μ g/m L, the MM plate on which the specimen had been spotted was placed in an incubator at 28 ℃ and incubated for 24h before observing the results.

The result analysis shows that AH L s is a diffusible small molecule, the more AH L s are contained in the reaction mixture, the larger the diameter of the blue ring appearing on the MM plate is, the strain has no degradation effect on AH L s, on the contrary, the less AH L s are contained in the reaction mixture, the smaller the diameter of the generated blue ring is, even no blue ring appears, and the strain with the best degradation effect on AH L s is screened according to the experimental result and named as the strain W-7.

2. Identification and phylogenetic analysis of strain W-7

(1) The colony morphology is characterized in that the strain W-7 is streaked on L B solid culture medium, is cultured for 24h in a 30 ℃ biochemical incubator at constant temperature, is cultured on a nutrient agar plate for 48h, and is light yellow, the colony is flat, the surface is smooth and translucent, the edge is neat, as shown in figure 1, the strain W-7 is diffusively turbid in L B liquid culture medium, is aerobic, and grows well at 30 ℃, as shown in figure 2, the bacterial colony is light yellow on a blood plate.

(2) Morphological characteristics of the thallus: as shown in FIG. 3, the bacterial cells were rod-shaped and had 1 polar flagellum.

(3) Physiological and biochemical characteristics: the strain W-7 is gram-negative bacteria, and has positive reaction in aerobic, catalase test, oxidase test, fluorescent pigment test, nitrate reduction test and nitrate gas production test, and negative reaction in hemolysis test, gelatin liquefaction test, hydrogen sulfide test and starch hydrolysis test; the optimum growth temperature is 30 ℃, and the optimum pH is 7.0. The physiological and biochemical identification results are shown in table 1.

(4)16S rDNA sequence and phylogenetic analysis: the length of the 16S rDNA gene sequence of the strain W-7 is 1411bp, and the strain W-7 is compared with an NCBI database (http:// www.ncbi.nlm.nih.gov /), and the strain W-7 and Pseudomonas nitroreducens have good homology (more than 99 percent) and the phylogenetic tree is shown in figure 4.

(5) Biolog automatic analysis system identification of microorganisms:

biolog microorganism automatic analysis is a microorganism identification method, data analysis is carried out through a computer, the identification process is rapid, and the result is accurate and reliable. Dis (distance) and sim (similarity) are 2 important parameters of the identification result, and represent the matching degree of the test result and the corresponding data of the database. When DIS is less than 5.0, the closer the SIM value is to 1, the higher the reliability of the identification result.

The result of the identification of the Biolog microorganism automatic analysis system of the strain W-7 shows that the strain W-7 is most similar to the Pseudomonas nitroreducens, the DIS is less than 5.0, and the SIM value is 0.741, which indicates that the strain W-7 is well matched with the Pseudomonas nitroreducens. The results of carbon source utilization by the strain W-7 are shown in Table 2.

In conclusion, the strain W-7 is identified as Pseudomonas nitroreducens (Pseudomonas nitroreducens) through morphological characteristics, physiological and biochemical characteristics, 16S rDNA gene sequence analysis and Biolog microorganism automatic analysis system identification of the strain W-7, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m2017649, the preservation address is Wuhan university in China.

TABLE 1 physiological and biochemical identification results of the strain W-7

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

TABLE 2 identification of the carbon Source utilization by Strain W-7

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

Example 2 antibiotic susceptibility analysis of Strain W-7

In order to be able to better study the biocontrol potential of the strain W-7 obtained in example 1, we have conducted intensive studies on the biological properties of this strain W-7. The sensitivity of strain W-7 to different antibiotics was investigated experimentally, as shown in FIG. 5.

Experimental results show that the resistance of the strain W-7 to ampicillin reaches more than 400 mu g/m L, the resistance to gentamicin, streptomycin and chloramphenicol reaches more than 200 mu g/m L, and the resistance to tetracycline reaches 10 mu g/m L.

EXAMPLE 3 determination of the degrading ability of the Strain W-7 to OdDH L

1. Strain culture and sample collection, wherein strain W-7 is streaked on L B solid medium plate, cultured overnight at 30 ℃, single colony is picked, cultured overnight with L B liquid medium to obtain bacterial liquid, and 1 OD is taken₆₀₀The thalli with the value is inoculated into 1m L MSM inorganic salt culture medium with OdDH L as a unique carbon source, the final concentration of OdDH L in the MSM inorganic salt culture medium is 80 mu M, the reaction mixture is placed in a 2m L centrifuge tube, the centrifuge tube is cultured for 24 h.24h under the conditions of 30 ℃ and 200rpm, the reaction mixture with the value of 5 mu L is sampled to the top end of an agar strip, then a reporter strain (Agrobacterium tumefaciens NT1) bacterial liquid is sampled at the lower point, and then a culture dish with the reaction mixture and the reporter strain is placedThe agar strips are obtained by cutting MM plates with the concentration of 40 mu g/m L X-gal into strips.

Among experimental controls, DH5 α was a negative control, no degradation was observed for AH L s, B23 was a positive control, degradation was observed for AH L s Bacillus thuringiensis subsp. Israelensis B23 contains the gene encoding the degrading enzyme aiiA of AH L s, and degradation was observed for AH L s (Dong Y, Xu J, L i X, et al AiiA, enzyme which is inactivated against the enzyme and molecules of the acyl-amino-serine lactone, quorum-sensing signal and molecules of the viral enzyme of Erwinia carotovora [ J ]. Proc Natl Acad Sci USA, 2000, 97 (7): 3526-3531.). the experimental results show (FIG. 6), the experimental results of both CK and DH5 α groups do not contain the reporter strain, and the residual amounts of the two experimental strains in the experimental group are reported to be identical, and the experimental results of the two experimental groups are reported for the complete degradation of DH 4837, the experimental group, the experimental results are reported for the same strain, and the experimental group for the experimental results of the experimental group are reported for the same reaction of DH 4837, the experimental results, the experimental group for the residual amounts of the experimental strains of the experimental group, the experimental results are reported for the experimental group, and the experimental group for the experimental results are reported for the experimental group for the experimental results.

EXAMPLE 4 determination of OdDH L relationship curves for growth and degradation of Strain W-7

1. Selecting single bacterial colony of strain W-7, inoculating to L B culture medium, pre-culturing to logarithmic phase, taking 1 OD thallus, re-suspending the thallus in MSM basic culture medium, adding the re-suspended liquid into 19m L MSM basic culture medium, adding OdDH L mother liquor to make its final concentration be 0.2mM, culturing at 30 deg.C and 200rpm, timing sampling, collecting samples at different time points and determining OD₆₀₀The value represents the growth of the strain W-7, and the residual amount of OdDH L measured with HP L C represents the degradation of OdDH L by the strain W-7.

2. HP L C determination method

HP L C: Waters 2695. chromatographic column: Kinetex EVO C18 reverse phase chromatographic column (250 μm × 4.6.6 mm × 5 μm) flow rate: 0.8m L/min column temperature: 30 ℃ mobile phase: methanol: water: 70; 30 (v: v) detection wavelength: 210 nm. sample injection: 20 μ L.

3. Results analysis, the HP L C test result is shown in FIG. 7, the graph A is a comparison graph of the non-inoculated strain W-7, the graph B, C, D, E is a degradation graph of the strain W-7 to OdDH L12 h, 24h, 36h and 48h respectively, the degradation rate reaches 63.6 percent,85.9%, 91.9%, 100%, corresponding to the OD of strain W-7 at that time₆₀₀The values are 0.054, 0.132, 0.162 and 0.165 respectively, a growth curve and a degradation curve graph are shown in fig. 8, degradation of OdDH L is in positive correlation with the growth of the strain, the strain has no detention period and rapidly enters a growth logarithmic phase in the presence of OdDH L, the strain degrades OdDH L fastest in 0-24 h, the strain is cultured for 48h, OdDH L is completely decomposed, and the natural degradation rate in OdDH L24 in the control is less than 40%.

The result shows that the pseudomonas nitroreducens W-7 have obvious and rapid degradation effect on the OdDH L, and have great application potential in the aspect of preventing and treating pathogenic bacteria harm mediated by the OdDH L.

Example 5 determination of DSF degrading Activity of Strain W-7

This example uses MM mineral salts medium with DSF as the sole carbon source to determine the DSF degrading activity of strain W-7.

1. Preparation of MM inorganic salt medium with DSF as sole carbon source: a DSF mother liquor (methanol as solvent, 100mM concentration) and MM mineral salts medium without any carbon source were prepared. Adding a certain amount of DSF mother liquor into an MM inorganic salt culture medium without any carbon source, uniformly mixing, pouring plates, and airing to ensure that the final concentration of DSF is 5 mM.

2. Inoculating strain W-7 frozen at-80 deg.C into L B liquid culture medium according to the volume ratio of 1: 100, culturing overnight at 30 deg.C and 200rpm, centrifuging the cultured bacterial liquid at 4000rpm for 10min, discarding supernatant, re-suspending the strain with MSM to obtain MSM re-suspension of the strain to be screened, spotting the MSM re-suspension of 0.5 μ L strain to be screened on MM inorganic salt culture medium plate with 5mM DSF as the only carbon source, and setting MSM and DH5 α experimental groups as controls.

3. And result analysis shows that as shown in FIG. 9, the MSM and DH5 α experimental groups have no hydrolysis ring, which indicates that MSM has no influence on the experiment, DH5 α can not degrade DSF. W-7 experimental group has obvious hydrolysis ring, which indicates that strain W-7 can degrade and use DSF as the only carbon source.

EXAMPLE 6 biocontrol Effect of Strain W-7 on Potato Soft rot disease

This example illustrates the use of the pathogen causing soft rot in potato, Dickeya zeae EC1(Hussain M B, Zhang H B, Xu J L, et al. the acyl-hormone binder-type plasmid-sensing systems cell motility and viruse of Erwinia chrysophanic pvzeae [ J ]. JBacteriol, 2008, 190 (3): 1045-1053) to study the biocontrol effect of strain W-7 on pathogenic bacteria that depend on AH L s.

1. Respectively streaking the strain W-7 and pathogenic bacteria dependent on AH L s, namely Dickeya zeae EC1 on L B solid medium plate, culturing overnight at 30 deg.C, respectively picking single colony, pre-culturing in L B medium to OD₆₀₀Setting W-7+ L B, DH5 α + L B, B23+ L B, EC1+ L B, W-7+ EC1, DH5 α + EC1, B23+ EC1 and seven experimental groups respectively to enable W-7, DH5 α, B23 and EC1 to have final working concentration OD₆₀₀In the experimental groups, DH5 α showed no degradation to AH L s, which was a negative control, B23 showed degradation to AH L s, which was a positive control, and 5. mu. L of the mixed bacterial suspension was inoculated to potatoes using a pipette.

2. As shown in FIG. 10, the results of the analysis show that the potatoes in the three experimental groups of W-7+ L B, DH5 α + L B and B23+ L B have no disease, which indicates that the potatoes in the three experimental groups of W-7, DH5 α and B23 have no pathogenicity to the potatoes, and the potato disease situations in the three experimental groups of EC1, DH5 α + EC1 and B23+ EC1 have no obvious difference.

Example 7 biocontrol Effect of Strain W-7 on cabbage Soft rot disease

In this example, the biocontrol effect of strain W-7 on pathogenic bacteria that depend on AH L S was studied, taking as an example the pathogenic bacteria Dickeya dadanti 3937(Glasner J D, Yang C, Recerchon S, et al. genome sequence of the plant-pathogenic bacteria Dickeya dadanti 3937[ J ]. J Bacteriol, 2011, 193 (8): 2076 and 2077.).

1. The strain W-7 and a pathogen relying on AH L s to cause diseases, namely Dickeya dadanti 3937 are streaked on L B solid medium plates respectively and cultured overnight at 30 ℃Single colonies were precultured to OD in L B medium₆₀₀L B, 3937, W-7, W-7+3937 and four experimental groups were set to obtain final working concentrations OD of 3937 and W-7, respectively₆₀₀Each experimental group was inoculated with 2 μ L of mixed inoculum using a pipette gun to a stem of cabbage.

2. As shown in FIG. 11, the result analysis shows that the inoculated parts of L B and W-7 two experimental groups have no disease on the inoculated Chinese cabbage stalks, which indicates that L B and W-7 have no pathogenicity on the Chinese cabbage stalks, compared with the inoculated part of the experimental group 3937, the inoculated parts of the experimental group W-7+3937 have no obvious soft rot symptom, and the experimental result shows that the strain W-7 has obvious biological control effect on the soft rot caused by Dickeya dadanti 3937.

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. A nitroreduction pseudomonas (Pseudomonas nitroreducens) strain W-7 capable of efficiently degrading microbial quorum sensing signal molecules is characterized in that the strain is preserved in China center for type culture Collection in 2017, 11 and 2 months, and the preservation number is CCTCC NO: m2017649.

2. Use of pseudomonas nitroreducens for degrading microbial quorum sensing signal molecules or for preparing products for degrading microbial quorum sensing signal molecules, wherein the pseudomonas nitroreducens is pseudomonas nitroreducens strain W-7 according to claim 1, and the microbial quorum sensing signal molecules are acyl homoserine lactone substances or DSF.

3. The use of Pseudomonas nitroreducens for controlling plant diseases which are pathogenic in a mediated manner by a microbial quorum sensing signal molecule, or for preparing a control agent for pathogenic bacteria which are pathogenic in a mediated manner by a microbial quorum sensing signal molecule, wherein the Pseudomonas nitroreducens is the Pseudomonas nitroreducens strain W-7 according to claim 1, and the microbial quorum sensing signal molecule is an acyl homoserine lactone substance or DSF.

4. A method for controlling pathogenic bacterial diseases which are pathogenic depending on microbial quorum sensing signals, which is characterized in that crops are treated by using bacterial suspension of nitropseudomonas reductans, wherein the nitropseudomonas reductans is the nitropseudomonas reductans strain W-7 in claim 1, and the microbial quorum sensing signal molecules are acyl homoserine lactone substances or DSF.

5. The method of claim 4, wherein the pathogenic bacteria that are pathogenic in dependence on the microbial quorum sensing signals are: erwinia (Erwinia), Dieckea (Dickeya), Xanthomonas (Xanthomonas) Burkholderia (Burkholderia) or Pseudomonas aeruginosa (Pseudomonas aeruginosa).

6. A degrading bacterial agent capable of degrading a quorum sensing signal molecule, comprising the strain W-7 and/or a bacterial suspension thereof according to claim 1.

7. A biocontrol agent for a pathogenic bacterium which causes a disease by a quorum sensing signal molecule, which comprises the strain W-7 and/or a suspension thereof according to claim 1.