CN108048351B

CN108048351B - Acylhomoserine lactone degrading bacterium and application thereof in disease control

Info

Publication number: CN108048351B
Application number: CN201711375319.2A
Authority: CN
Inventors: 陈少华; 范兴辉; 张炼辉; 叶田; 王惠杉; 郭云帆; 张译尹
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2017-12-19
Filing date: 2017-12-19
Publication date: 2021-02-19
Anticipated expiration: 2037-12-19
Also published as: CN108048351A

Abstract

The invention discloses an acyl homoserine lactone degrading bacterium and application thereof in disease control. The degrading bacteria are acinetobacter (A), (B), (C)Acinetobacter schindleri) In particular to an acinetobacter strain XJ-10 which is preserved in China center for type culture Collection in 2017 at 11/2, and the preservation number is CCTCC NO: m2017650. The strain can degrade quorum sensing signal molecules and grow by taking the quorum sensing signal molecules as a unique carbon source, and has an obvious and rapid degradation effect on the quorum sensing signal molecules of AHLs; therefore, quorum sensing signal molecules can be degraded to block the quorum sensing communication and inhibit the expression of pathogenic factors of pathogenic bacteria, so that the purpose of preventing and treating diseases is achieved, and the quorum sensing signal molecules have great application potential in the aspect of preventing and treating the harm of pathogenic bacteria which depend on AHLs mediated pathogenesis; but also can reduce the abuse problem of chemical pesticides and provide a new strategy for biologically preventing and controlling the harm of plant pathogenic bacteria.

Description

Acylhomoserine lactone degrading bacterium and application thereof in disease control

Technical Field

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

Background

Microorganisms sense changes in population density by monitoring the concentration of extracellular signaling molecules (auto-inducers), which, when they reach a certain threshold, initiate the expression of the target gene, coordinate the behavior of the population, namely Quorum Sensing (QS) (Whiteley M, Diggle S P, Greenberg E P. progress in and progress of bacterial Quorum Sensing research [ J ]. Nature.2017,551(7680): 313-) 320.). Quorum sensing is widely present in microbial populations and is associated with various biological functions of microorganisms, such as: bioluminescence, symbiosis, biofilm formation, antibiotic synthesis, population mobility, plasmid transfer, sporulation, gene exchange, etc. (Davies DG, Parsek MR, Pearson JP, et al. the innovative of cell-to-cell signals in the later of a bacterial biology [ J ] Science,1998,280(5361): 295-.

The Acyl Homoserine Lactones (AHLs) are quorum sensing signals specific to gram-negative bacteria, and most of the substances have the same cyclic structure of homoserine lactone and have Acyl side chains, but the lengths and the saturations of the Acyl side chains are different. For example, the conventional N- (3-oxodecanoyl) -L-homoserine lactone (OdDHL) and N- (3-oxohexanoyl) -L-homoserine lactone (OHL) belong to the AHLs, and the difference is that the acyl side chain length is different. AHLs signals are widely present in gram-negative bacteria, including the plant pathogens Erwinia (Erwinia), dicamba (Dickeya), Pseudomonas aeruginosa (Pseudomonas aeruginosa), etc., and are associated with pathogenic bacteria, such as: OHL is a quorum sensing signal molecule of plant pathogenic bacteria Dickeya zeae EC1, and which mediate quorum sensing phenomena and are associated with pathogenicity (Hussain M B, Zhang H B, Xu J L, et al. the acyl-homolog lactone-type quorum-sensing systems, ll motility and virus of Erwinia chrysogenm pv. zeae [ J ]. Journal of Biotechnology,2008,190(3):1045 and 1053.), OdDHL is a quorum sensing signal molecule of the human pathogen Pseudomonas aeruginosa, and its mediated quorum sensing system forms an immediate relationship with the biofilm (Huang J, Han J I, Zhang L H, et al. the inactivation of acyl-homolog allergen polysaccharide kinase, polysaccharide J, 9J, 5925, 5969, P5925, P.A).

Quorum sensing Quenching (QQ), a mechanism that interferes with the Quorum sensing system by inhibiting the synthesis, accumulation, monitoring, or enzymatic degradation or modification of signal molecules (Fetzner S. Quorum sequencing enzymes [ J ]. Journal of Biotechnology,2015,201: 2-14.). 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. Quorum sensing quenching is a new way for effectively preventing and controlling 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 invention aims to overcome the defects and shortcomings of the existing phytopathogen control technology and provide a strain capable of efficiently degrading quorum sensing signal molecules AHLs (AHLs-like metabolites) and AHLs analogue and application thereof as biocontrol bacteria.

The invention aims to provide an Acinetobacter (Acinetobacter schendleri) strain XJ-10 capable of degrading AHLs quorum sensing signal molecules.

The invention also aims to provide application of the acinetobacter in prevention and treatment of plant diseases dependent on AHLs mediated pathogenesis.

It is still another object of the present invention to provide a method for controlling pathogenic bacterial diseases that depend on AHLs.

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

an Acinetobacter (Acinetobacter schindleri) strain XJ-10 capable of degrading AHLs quorum sensing signal molecules is preserved in China Center for Type Culture Collection (CCTCC) in 11 and 2 months in 2017, and the preservation number is CCTCC NO: m2017650, the preservation address is: wuhan, Wuhan university, China.

The strain is obtained by artificially screening, separating and purifying a soil sample collected from Xinjiang, and is identified to be Acinetobacter (Acinetobacter schindleri) and named as XJ-10 through morphological characteristics, physiological and biochemical characteristics and 16S rDNA phylogenetic analysis of the strain.

Meanwhile, different antibiotic sensitivity analyses are carried out on the strain XJ-10. The resistance of the strain XJ-10 to ampicillin reaches more than 400 mug/mL, the resistance to kanamycin reaches 200 mug/mL, the resistance to gentamicin reaches 100 mug/mL, and the resistance to streptomycin and chloramphenicol reaches 20 mug/mL.

The Acinetobacter (Acinetobacter schindleri) strain XJ-10 provided by the invention can be used for rapidly degrading quorum sensing signal molecules AHLs. The acinetobacter can degrade gram-negative flora quorum sensing signals, especially microorganism quorum sensing signals such as Erwinia (Erwinia), Dickaya (Dickeya), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and the like. The signal molecules of the pathogenic bacteria of the gram-negative group are homoserine lactones.

In the application of degrading quorum sensing signal molecules, experimental research shows that the Acinetobacter (Acinetobacter schendleri) strain XJ-10 has obvious and rapid degradation effect on quorum sensing signal molecules AHLs. Can grow normally in a culture medium with OdDHL as a sole carbon source, and can completely decompose quorum sensing signal molecule OdDHL with an initial concentration of 0.2mM within 48 h. Meanwhile, the strain has a good degradation effect on quorum sensing signal molecules OHL. Has great application potential in preventing and treating AHLs mediated pathogenic bacteria harm.

Therefore, the application of the acinetobacter in degrading quorum sensing signal molecules AHLs and/or AHLs signal analogues or preparing products for degrading AHLs and/or AHLs signal analogues; the application in preventing and treating plant diseases which depend on AHLs to mediate pathogenicity or the application in preparing a preventing and treating preparation of pathogenic bacteria which depend on AHLs to cause pathogenicity; are intended to be within the scope of the present invention.

Specifically, the AHLs signaling species is a homoserine lactone species. Including OdDHL and OHHL, etc.

Preferably, the Acinetobacter is the above Acinetobacter strain XJ-10.

In addition, based on the application, the Acinetobacter (Acinetobacter schendleri) strain XJ-10 provided by the invention has the development potential of biological control.

The invention also provides a method for preventing and treating pathogenic bacteria diseases depending on AHLs, in particular to a method for treating crops by using the bacterial suspension of acinetobacter. The treatment mode is inoculation or spraying treatment.

Preferably, the Acinetobacter is the above Acinetobacter strain XJ-10.

Wherein the pathogenic bacteria which depend on AHLs for pathogenesis comprise: dicarba (Dickeya), Erwinia (Erwinia) or Pseudomonas aeruginosa (Pseudomonas aeruginosa), etc.; the acinetobacter strain XJ-10 has a remarkable biocontrol effect on diseases of pathogenic bacteria depending on AHLs.

Preferably, the most suitable culture medium for preparing the bacterial suspension of the strain XJ-10 is a nutrient-rich culture medium, and the formula of the nutrient-rich culture medium is as follows: 10.0g/L of tryptone, 5.0g/L of yeast extract, 10.0g/L of sodium chloride, pH 6.8-7.2 and sterilization at 121 ℃ for 15-25 min.

In addition, a degrading bacterial agent containing the strain XJ-10 and/or the bacterial suspension thereof and capable of degrading the quorum sensing signal molecules AHLs and a biocontrol agent containing the strain XJ-10 and/or the bacterial suspension thereof and capable of resisting pathogenic bacteria causing AHLs are also within the protection scope of the invention.

The invention has the following beneficial effects:

the research of the invention finds that the acinetobacter has better degradation activity aiming at the quorum sensing signal molecules AHLs, has stable, obvious and quick degradation effect, and provides a new way and method for preventing and treating pathogenic bacteria mediated by the AHLs.

Meanwhile, the invention also provides an acinetobacter strain XJ-10 with obvious rapid degradation activity on quorum sensing signal molecules, which has great application potential in the aspect of preventing and treating pathogenic bacteria mediated by AHLs, 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.

Drawings

FIG. 1 is a colony morphology of the strain XJ-10 of the present invention on LB medium.

FIG. 2 is a colony morphology of strain XJ-10 of the present invention on blood plating medium.

FIG. 3 is a scanning electron micrograph of the strain XJ-10 of the present invention.

FIG. 4 is a phylogenetic tree analysis diagram of strain XJ-10 of the present invention.

FIG. 5 is a graph showing the growth of strain XJ-10 of the present invention in various antibiotics.

FIG. 6 is a graph showing the measurement of the degrading activity of the strain XJ-10 of the present invention.

FIG. 7 is an HPLC chart showing the degradation of OdDHL by the strain XJ-10 of the present invention (FIG. A is a chart showing the comparison of the non-inoculated strain XJ-10, and FIG. B, C is a high performance liquid chromatogram showing the degradation of OdDHL by the strain XJ-10 at 12h and 24h, respectively).

FIG. 8 shows the disease onset of the strains XJ-10, DH5 alpha, B23, EC1 of the present invention inoculated individually and together with EC1 inoculated into potato tuber for 30 h.

FIG. 9 shows the onset of the bacterial strains XJ-10 and 3937 of the present invention after they were inoculated separately and together to the stem of Chinese cabbage for 24 hours.

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 present invention are commercially available.

The materials used in the following examples are as follows:

LB culture medium: 10.0g/L of tryptone, 5.0g/L of yeast extract, 10.0g/L of sodium chloride, pH 6.8-7.2 and sterilization at 121 ℃ for 15-25 min. The LB solid medium formulation is to add 1.5% (w/v) agar to the liquid medium.

Basal salt medium (MSM): (NH4)₂SO₄，2.0g/L；CaCl₂·2H₂O，0.01g/L；Na₂HPO₄·12H₂O，1.5g/L；KH₂PO₄，1.5g/L；MgSO₄·7H₂O，0.2g/L；FeSO₄·7H₂O，0.001g/L；pH 6.5。

Basal medium (MM): k₂HPO₄，10.5g/L；KH₂PO₄，4.5g/L；(NH4)₂SO₄，2.0g/L；Mannitol，2.0g/L；Glycerol，2.0g/L；MgSO₄·7H₂O，0.2g/L；CaCl₂，0.01g/L；

FeSO₄，0.005g/L；MnCl₂，0.002g/L；pH 6.5。

OdDHL and OHL were purchased from Shanghai Youder chemical science and technology Co., Ltd, and X-gal and reagents required by a culture medium were purchased from Guangzhou Qixiang, Huaqisheng, and the like.

Example 1 isolation and screening of OdDHL-degrading bacterium (Acinetobacter strain XJ-10)

1. Isolation and screening of Acinetobacter strain XJ-10

(1) Soil sample collection: soil samples collected from Xinjiang were used as the source of the microorganisms.

Soil samples were collected from farmlands in Xinjiang province 10, 9/2014, sampled, bagged, stored as microbial sources and brought back to the laboratory for strain isolation and screening.

(2) Enrichment culture of the strain: preparing a basal salt medium (MSM), filling 50mL of MSM medium into a 250mL triangular flask, sterilizing at 121 ℃ for 15-25 min, cooling, adding OdDHL mother solution (methanol as a solvent) under aseptic conditions to make the final concentration of OdDHL 5 muM, adding 5g of soil sample, placing the mixture at 30 ℃ and culturing for 7d in a shaking table at 200rpm, and transferring the mixture into the MSM medium with the concentration of OdDHL 10 muM according to the inoculum concentration of 10%. After culturing for 7 days under the same conditions, the cells were inoculated into MSM medium with an OdDHL concentration of 20. mu.M in an amount of 10%, and then cultured for 7 days under the same conditions. By analogy, the concentration of OdDHL was increased continuously.

(3) Strain separation: the isolation of the strains was carried out by plate coating.

Taking 1mL of final MSM culture medium fermentation broth, diluting with sterile water to 10 concentration^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8Then 100 mul of fermentation liquor with various concentrations is respectively sucked, evenly coated on an LB solid plate, dried and cultured for 24h at 30 ℃. Picking sheets of different shapes growing on the flat plateThe colony is repeatedly streaked and cultured on an LB solid plate for purification until a single strain is separated. The single strain was stored at-80 ℃ by glycerol conservation for further testing for degradation activity.

(4) Strain screening: the strain having the degrading activity was selected using a reporter strain (Agrobacterium tumefaciens NT 1).

Activating the strain to be screened from-80 ℃, streaking on an LB solid medium flat plate, and culturing for 24h in an incubator at 30 ℃. And (3) selecting a single colony, inoculating the single colony to an LB liquid culture medium, and culturing overnight at the temperature of 30 ℃ and the speed of 200rpm to obtain a bacterial liquid. Take 1OD₆₀₀The cells were resuspended in 1mL of MSM medium containing OdDHL as the sole carbon source to obtain a mixture to be cultured, and the concentration of OdDHL in the MSM medium was 20. mu.M. The mixture to be incubated was transferred to 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 MM plates coated with 200. mu.L of reporter strain broth, the OD of which₆₀₀MM plates contained X-gal at a concentration of 40 μ g/mL ═ 0.4. The MM plates on which the samples had been spotted were placed in an incubator at 28 ℃ and the results of the experiment were observed after 24 h.

The result analysis shows that the OdDHL has diffusivity, the more the OdDHL contained in the reaction mixture, the longer the diffusion distance from the sample point to the periphery is, the larger the radius of the blue circle appeared on the MM plate is, and the closer the radius of the blue circle is to that of the CK experimental group, the weaker the degradation effect of the strain on the OdDHL is or the degradation effect is not shown, and on the contrary, the less the OdDHL contained in the reaction mixture is, the smaller the diameter of the generated blue circle is or even the blue circle is not appeared. And (3) screening out a strain with the best degradation effect on the OdDHL according to the experimental result, and naming the strain as degrading bacteria XJ-10.

Example 2 identification of Acinetobacter Strain XJ-10

In this example, morphological characteristics, physiological and biochemical characteristics, and developmental analysis of 16S rDNA system were performed on the degrading strain XJ-10, and the strain was identified as Acinetobacter (Acinetobacter schendleri). The method comprises the following specific steps:

(1) and (3) colony morphology characteristics: culturing on LB solid plate for 48h, the colony is flat, the surface is smooth and opaque, and the edge is neat, as shown in FIG. 1; the bacterial colonies appeared off-white on the blood plates, as shown in FIG. 2; the strain showed diffusible turbidity in LB liquid medium, and grew well under aerobic conditions at 30 ℃.

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

(3) Physiological and biochemical characteristics: the strain is gram-negative bacteria, aerobic, positive in catalase test reaction, and negative in oxidase, hemolytic test, gelatin liquefaction and starch hydrolysis test reaction; the range of the growth temperature is 20-41 ℃, the optimum growth temperature is 30 ℃, and the optimum pH is 7.0. The physiological and biochemical identification results are shown in tables 1 and 2.

(4)16S rDNA sequence and phylogenetic analysis: the length of the 16S rDNA gene sequence of the strain XJ-10 is 1420bp, and the strain XJ-10 has higher homology with Acinetobacter schindleri after being compared with NCBI database (http:// www.ncbi.nlm.nih.gov /), and the phylogenetic tree is shown in figure 4.

In summary, the morphological characteristics, physiological and biochemical characteristics and 16S rDNA gene sequence of the strain XJ-10 are identified and analyzed, the strain is identified to belong to Acinetobacter (Acinetobacter schendleri) and is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m2017650, wherein the preservation address is Wuhan university in Wuhan, China.

TABLE 1 physiological and biochemical identification results of Strain XJ-10

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

TABLE 2 identification of the carbon Source utilization of Strain XJ-10

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

Example 3 antibiotic susceptibility analysis of Strain XJ-10

To enable better study of strain XJ-10, this example carried out a number of antibiotic susceptibility tests on strain XJ-10.

And (3) analyzing an experimental result: as shown in FIG. 5, the strain XJ-10 has ampicillin resistance of 400. mu.g/mL or more, kanamycin resistance of 200. mu.g/mL, gentamicin resistance of 100. mu.g/mL, and streptomycin and chloramphenicol resistance of 20. mu.g/mL. This result is useful for reference in subsequent studies to select suitable antibiotics.

EXAMPLE 4 Activity assay of Strain XJ-10 for degradation of OdDHL

This example uses a reporter strain (Agrobacterium tumefaciens NT1) to test the effect of strain XJ-10 on the degradation of OdDHL.

1. The experimental steps are as follows:

the strain XJ-10 was activated by plating with LB solid medium, and the plate was incubated overnight in an incubator at 30 ℃. A single colony was picked and inoculated into liquid LB medium, and cultured overnight at 30 ℃ and 200rpm to obtain a bacterial solution. Take 1OD₆₀₀Taking 1mL of MSM culture medium with OdDHL as a sole carbon source to resuspend the thalli to obtain a reaction mixture, wherein the concentration of OdDHL in the MSM culture medium is 80 mu M. The reaction mixture was transferred to 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 the agar strip, followed by spotting the reporter strain (Agrobacterium tumefaciens NT1) on the bottom in sequence. The agar strips with the reaction mixture and the reporter strain spotted thereon were then placed in an incubator at 28 ℃ and incubated for 24 hours before observing the results. Wherein the agar strips are obtained by cutting MM plates with the concentration of 40 mug/mL X-gal.

Escherichia coli DH5 alpha is a strain known to have no degrading effect on OdDHL, and is a negative control group in the present example; bacillus thuringiensis subsp. Israelensis B23 is a strain known to have a degrading effect on OdDHL, and in this example is a positive control (Dong Y, Xu J, Li X, et al AiiA, an enzyme that inactivates the acyl hormone amino acid-sensing signal and enzymes the virus of Erwinia carotovora [ J ]. Proceedings of the National Academy of Sciences,2000,97(7):3526 @); CK is blank control without bacteria.

2. And (4) analyzing results:

the results are shown in FIG. 6, and the distances from the top of agar strip to the last blue point of the reporter strain in the CK and DH5 alpha experimental groups are basically consistent, which indicates that the OdDHL content in the reaction mixture of the two experimental groups is similar and is consistent with the fact that DH5 alpha has no OdDHL degradation capability. Neither of the B23 and XJ-10 experimental groups reported that the strains did not turn blue, indicating that the reaction mixtures of both experimental groups did not contain OdDHL, which had been completely degraded, i.e., XJ-10 was able to degrade OdDHL.

Example 5 Activity of Strain XJ-10 for degrading OdDHL Using HPLC

1. The method comprises the following steps: activating a strain XJ-10 frozen at-80 ℃ by using an LB solid plate, culturing at 30 ℃ for 24h, selecting a single colony on the plate, inoculating the single colony on a liquid LB culture medium, and culturing overnight at 30 ℃ and 200rpm to obtain a bacterial liquid. 1OD cell was taken, and the cell was resuspended in 1mL of MSM medium, and the resuspended solution was added to 19mL of MSM basal medium, and OdDHL mother liquor (methanol as solvent) was added to the medium to give a final concentration of 0.2 mM. Culturing at 30 ℃ and 200rpm, sampling at three time points of 0h, 12h and 24h, extracting residual OdDHL in the sample, and determining the residual amount of OdDHL by HPLC to show the degradation of OdDHL by strain XJ-10.

2. The extraction method of OdDHL in the sample comprises the following steps: taking a proper amount of sample into a 15mL centrifuge tube, centrifuging for 5min at the speed of 4000rpm, taking 5mL supernatant into a 50mL separating funnel, adding equal volume of ethyl acetate into the separating funnel, violently shaking for 3min, standing, layering, transferring the lower layer solution into the 15mL centrifuge tube, filtering the upper layer solution into a 50mL round-bottom flask through a funnel, and paving filter paper in the funnel. The lower solution was extracted 1 more time as described above. The filtrate was combined into a round-bottomed flask, evaporated to dryness by rotary evaporation at a constant temperature of 45 ℃, the round-bottomed flask was washed with chromatographic methanol 2 times, and the volume was made to 2mL, and the solution was filtered through a 0.45 μ M organic filter membrane into a sample bottle, and the residual amount thereof was measured by HPLC.

3. Method for determining OdDHL residual amount by HPLC:

HPLC instrument model: waters 2695. A chromatographic column: c₁₈Reversed phase chromatography column (250. mu. m.times.4.6 mm. times.5 μm). The flow rate was 0.8 mL/min. The column temperature was 30 ℃. Mobile phase: methanol: 70 parts of water; 30 (v: v). The detection wavelength was 210 nm. The amount of sample was 20. mu.L.

4. The OdDHL degradation rate calculation method comprises the following steps: percent degradation (%) - (1-A)₁/A₀)×100，A₁To degrade the residual concentration of OdDHL after bacterial treatment, A₀Is the residual concentration of OdDHL after control treatment.

5. And (3) analyzing an experimental result:

the HPLC detection result is shown in FIG. 7, wherein the A is a comparison chart of the non-inoculated strain XJ-10, the B, C is a degradation chart of the strain XJ-10 to OdDHL 12h and 24h, the degradation rates reach 85.71% and 98.8%, respectively, and the OD of the strain XJ-10 at the corresponding time is corresponding to the degradation rate of the strain XJ-10₆₀₀The values are 0.144 and 0.233, respectively. Experiments show that under the condition that OdDHL exists as a unique carbon source, the strain can rapidly degrade the OdDHL and utilize the OdDHL to grow.

EXAMPLE 6 biocontrol Effect of Strain XJ-10 on Potato Soft rot disease

In this example, the biocontrol effect of strain XJ-10 on pathogenic bacteria that depend on AHLs was studied, taking as an example the plant soft rot pathogen Dickeya zeae EC1(Hussain M B, Zhang H B, Xu J L, et al, the acyl-hormone emulsion-type quantitative-sensing systems cell mobility and vision of Erwinia chrysogeni pv. zeae [ J ]. Journal of Biotechnology,2008,190(3): 1045-.

1. The method comprises the following steps:

the strain XJ-10 and EC1 were activated by LB solid medium plate and cultured at 30 ℃ for 24 hours. After 24 hours, single colonies on the plates were picked, inoculated into liquid LB medium, and cultured overnight at 30 ℃ and 200 rpm. Seven experimental groups were set up: XJ-10+ LB, DH5 α + LB, B23+ LB, EC1+ LB, XJ-10+ EC1, DH5 α + EC1, B23+ EC 1.

Among them, Escherichia coli DH 5. alpha. is a strain known to have no degrading effect on OdDHL, and is a negative control group in this example. Bacillus thuringiensis subsp israelensis B23 is a strain known to have a degrading effect on OdDHL, and is a positive control in this example. In the experiment, XJ-10, DH5 alpha, B23 and EC1 final working solution OD₆₀₀1.0. Each experimental group was inoculated with 5. mu.L of mixed broth by pipette gun into the center of potato slices.

2. And (4) analyzing results:

as shown in FIG. 8, the potatoes in the three experimental groups of XJ-10+ LB, DH5 alpha + LB and B23+ LB have no disease, which indicates that XJ-10, DH5 alpha and B23 have no pathogenicity to the potatoes; the disease conditions of the potatoes in three experimental groups of EC1, DH5 alpha + EC1 and B23+ EC1 are not obviously different. The severity of the disease of the potatoes in the experimental group XJ-10+ EC1 is obviously lower than that of the potatoes in the three experimental groups of EC1, DH5 alpha + EC1 and B23+ EC 1. Experimental results show that the strain XJ-10 has a remarkable biocontrol effect on soft rot caused by Dickeya zeae EC 1.

EXAMPLE 7 biocontrol Effect of Strain XJ-10 on cabbage Soft rot disease

In this example, the biocontrol effect of strain XJ-10 on AHLs-dependent pathogenic bacteria was studied, using as an example the pathogen Dickeya dadanii 3937(Glasner J D, Yang C, Reversan S, et al genome sequence of the plant-pathogenic bacteria Dickeya dadanii 3937[ J ]. Journal of Biotechnology,2011,193(8):2076 and 2077), which is capable of causing soft rot in cabbage.

1. The method comprises the following steps:

the strain XJ-10 and 3937 were activated by LB solid medium plate and cultured at 30 ℃ for 24 hours. After 24 hours, single colonies on the plates were picked, inoculated into liquid LB medium, and cultured overnight at 30 ℃ and 200 rpm. Respectively arranging LB, 3937, XJ-10, XJ-10+3937 and four experimental groups to ensure that the final working concentrations OD of 3937 and XJ-10 are₆₀₀1.0. Each experimental group was inoculated with 2. mu.L of the mixed inoculum to the cabbage stalk using a pipette gun. After 24hThe disease condition was examined.

2. And (4) analyzing results:

as shown in FIG. 9, no disease was observed in the inoculated portions of both experimental groups LB, XJ-10, which were inoculated and cultured for 24 hours, indicating that LB, XJ-10 were not pathogenic to the stem of Chinese cabbage. Compared with the inoculation position of the experimental group 3937, the inoculation position of the experimental group XJ-10+3937 has no obvious soft rot symptom and has a light degree of disease. Experimental results show that the strain XJ-10 has a remarkable biocontrol effect on soft rot caused by Dickeya dadantii 3937.

Claims

1. An Acinetobacter (Acinetobacter schindleri) strain XJ-10 capable of degrading acyl homoserine lactone substances is characterized in that the strain is preserved in China center for type culture Collection in 11 and 2 months in 2017, and the preservation number is CCTCC NO: m2017650.

2. Use of the acinetobacter strain XJ-10 according to claim 1 for degrading acyl homoserine lactones or for preparing products for degrading acyl homoserine lactones.

3. Use of the acinetobacter strain XJ-10 according to claim 1 for controlling diseases of plants which are pathogenic depending on acyl homoserine lactone-type substances, or for preparing a control preparation for pathogenic bacteria which are pathogenic depending on acyl homoserine lactone-type substances.

4. A method for controlling pathogenic diseases which are pathogenic depending on acyl homoserine lactones, characterized in that a crop is treated with a suspension of acinetobacter strain XJ-10 according to claim 1; the pathogenic bacteria which depend on the acyl homoserine lactone substances for disease are as follows: dieckea (Dickeya), Pseudomonas aeruginosa (Pseudomonas aeruginosa).

5. A degrading bacterial agent capable of degrading acyl homoserine lactone, which comprises Acinetobacter strain XJ-10 and/or a bacterial suspension thereof according to claim 1.

6. A biocontrol agent for pathogenic bacteria which depend on acyl homoserine lactones comprising Acinetobacter strain XJ-10 and/or a bacterial suspension thereof according to claim 1.