CN110713945B - Bacteroides nicotinovorans and application thereof in disease control - Google Patents

Abstract

The invention discloses a bacteroides nicotinovorans and an application thereof in disease control. The microbial quorum sensing signal quenched and sterilized bacteroides nicotinovorans (Paenarthrobacter nicotinovorans) strain D-9 obtained by screening is preserved in Guangdong province microbial strain collection center in 2019, 6 and 14 days, and the preservation number is GDMCC No. 60691. The strain has the activity of quenching quorum sensing signal molecules of N-acylhomoserine lactones (AHLs), and the quenching effect is stable and obvious, so that the strain has great application potential in preventing and treating pathogenic bacteria hazards which depend on AHLs quorum sensing signal mediated pathogenicity, such as carrot soft rot pectobacterium carotovorum subspecies (Pcc). The anti-pathogenicity drug taking colony quenching as an action mode has wide application prospect.

Description

Bacteroides nicotinovorans and application thereof in disease control

Technical Field

The invention belongs to the technical field of biological control. More particularly, relates to a bacteroides nicotinovorans and an application thereof in disease control.

Background

The cruciferous vegetables are various in types, mainly comprise Chinese cabbages, small green vegetables, cabbages, radishes, cauliflowers and the like, and have a large share in the vegetable sales market in China. Soft rot, virus disease and downy mildew are three major diseases of cruciferous vegetables worldwide (Yan bang, fan shan. comprehensive control of soft rot of cruciferous vegetables [ J ]. Sichuan agricultural science 2001,11: 32). The soft rot not only harms cruciferous vegetables, but also infects lettuce, potatoes, tomatoes, eggplants, hot peppers, cucumbers, carrots, celery, shallots, kiwi fruits and other crops, but the Chinese cabbage, radish and cabbage are the most seriously damaged, so that serious economic loss is caused. The yield of the Chinese cabbage is reduced by over 50 percent in the years with serious soft rot, even the Chinese cabbage is not harvested in pieces; the loss is aggravated by rot caused during transportation, sale and storage (Liu Shing. cruciferous vegetable soft rot and black rot [ J ]. New agriculture, 2002,8: 38-39). The main areas for planting celery in Beijing are Daxing, Shunyi and Tongzhou counties, wherein the fruit village in the Tongzhou county is a village for professionally producing celery, and the village once becomes the main production area of high-quality celery. However, in recent years, the celery planting process has the occurrence of the soft rot with strong destructiveness, which causes huge economic loss, serious disaster areas and even dead delivery (jin Zhi Wen, Song dynasty, xi scholar, etc.. separation and identification of the pathogen of the celery bacterial soft rot [ J ]. plant pathology reports, 2016,3: 304-. Kiwi fruit is known as one of the most popular fruits because of its rich nutrition and high medicinal value. The soft rot of kiwi fruit is the most serious disease in storage period. The disease has short course and is difficult to control, and brings great threat to the kiwi fruit industry (Zhouqu. research on etiology of kiwi fruit soft rot [ D ]. Sichuan university of agriculture, 2016).

The main pathogenic bacteria of the soft rot disease are pectobacterium carotovorum, which are important pathogenic bacteria of bacterial soft rot diseases of various economic crops. The species Pectobacterium carotovorum subsp. carotovora, Pcc and the species Pectobacterium carotovorum nigrosporus, Pca, belong to this species and can cause soft rot. Pcc caused bacterial soft rot has wide occurrence range, is generated in many countries and has serious harm, and has become a worldwide disease. Pcc can synthesize and secrete hydrolytic enzymes (pectinase, polygalacturonase, and protease) to break down plant cell wall, destroy plant tissue structure, and obtain water and nutrients to support self growth and reproduction. Pcc the synthesis and secretion of hydrolase is controlled by quorum sensing system, Pcc itself can produce and secrete one or more chemicals, when the population density becomes higher, the concentration of the chemical or chemicals is increased, after the concentration of the chemical or chemicals reaches a certain threshold, the gene for synthesizing and secreting Pcc hydrolase starts to express, so that Pcc has pathogenicity. These chemicals are known as quorum sensing signal molecules or Autoinducers (AIs) (Whiteley M, Diggle S P, Greenberg E P. progress in and program of bacterial quorum sensing research [ J ] Nature,2017,551:313-320.) Pcc, N- (3-oxohexanoyl) -L-homoserine lactone (OHL) in N-acylhomoserine lactones (AHLs).

The chemical pesticides such as benziothiazolinone, thifluzazole, amobam, streptomycin sulfate and zhongshengmycin are used for preventing and treating bacterial soft rot. However, the use of chemical pesticides in large quantities has brought about a series of serious problems known as environmental pollution, disruption of ecological balance, resistance to pathogenic bacteria and food safety, and in addition, the abuse of antibiotics has caused the development of microbial resistance. Therefore, a new, effective, least toxic disease control strategy, Quorum Quenching (QQ), was discovered. Quorum sensing quenching refers to the interference of a quorum sensing system through a mechanism of inhibiting the synthesis, accumulation and monitoring of signal molecules or carrying out enzymatic degradation or modification on the signal molecules, the inhibition of the expression of genes related to pathogenicity of microorganisms, and the weakening of the pathogenicity of the microorganisms, so that the aim of preventing and treating diseases is fulfilled.

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. As shown in the previous research of the inventor team, the (201711248767.6) nitroreduction Pseudomonas (Pseudomonas nitroreducens) has better function of microbial quorum sensing signal molecules, and has great popularization and application potential in the aspects of quenching direction of the quorum, inhibiting soft rot diseases and preventing and treating pathogenic bacteria harm which depends on microbial quorum sensing signal mediated pathogeny. More and more excellent microbial degradation bacterium libraries are enriched and constructed, and the method has great significance for preventing and treating pathogenic bacterium hazards depending on induction signals of microbial organisms to mediate diseases.

Disclosure of Invention

The invention aims to provide a novel microbial quorum sensing signal molecule DSF/AHLs quenching bacterium with better degradation effect, namely a Bacteroides nicotinovorans (Paenerthribacter nicotinovorans) strain D-9 and application thereof in biological control for solving crop diseases. The strain has obvious and rapid degradation effect on various AHLs quorum sensing signal molecules, has great application potential in the aspect of preventing and treating pathogen harm mediated by quorum sensing signal molecules, and provides a new development approach 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 selective pressure.

The invention aims to provide a bacteroides nicotinovorans (Paenarthrobacter nicotinovorans) strain D-9 capable of degrading microbial quorum sensing signal molecules.

Another object of the present invention is the use of Bacteroides nicotinovorans in the quenching of quorum sensing signaling molecules.

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

the research of the invention discovers that the bacteroides nicotinovorans (Paenarthrobacter nicotinovorans) has a very high-efficient effect of degrading microbial quorum sensing signal molecules, and a high-efficient degrading bacterium bacteroides nicotinovorans strain D-9 is obtained by screening, is preserved in Guangdong province microbial strain collection center in 2019, 6 and 14 days, has the preservation number of GDMCC No.60691 and the preservation address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

The strain is obtained by separating, purifying and screening soil samples collected from a tree garden and an orchid greenhouse of southern China university, the 16S rDNA sequence and morphological characteristics of the strain are analyzed, and the strain D-9 is identified as Bacteroides nicotinovorans (Paenarthrobacter nicotinovorans).

The colony morphology of the strain D-9 is characterized in that: the bacterial colony is white, round, convex, opaque, neat in edge and smooth in surface after being cultured on a nutrient agar plate for 24 hours.

The morphological characteristics of the thalli observed by an electron microscope are as follows: the cells are rod-shaped.

The strain D-9 shows high-concentration sensitivity to gentamicin (gen) and carbenicillin (carb), and low-concentration resistance to 40 mg/mL; is highly sensitive to ampicillin (amp), kanamycin (kan), streptomycin (str) and Tetracycline (TC), and shows no resistance regardless of the concentration.

Experimental results show that the Bacteroides nicotinovorans strain D-9 has quenching activity on quorum sensing signal molecules of the AHLs of the middle and long chains, and can obviously and rapidly degrade the AHLs of the middle and long chains. Strain D-9 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 application of Bacteroides nicotinovorans in quenching quorum sensing signal molecules AHLs or in preparing products for degrading AHLs also belongs to the protection scope of the invention.

The application of the bacteroides nicotinovorans in preventing and treating the plant diseases which are pathogenic mediated by the quorum sensing signal molecules AHLs or the application in preparing the preventing and treating preparation of the pathogenic bacteria which are pathogenic depending on the quorum sensing signal molecules AHLs also belongs to the protection scope of the invention.

Preferably, in any of the above applications, the Bacteroides nicotinovorans is Bacteroides nicotinovorans strain D-9.

A method for preventing and treating the pathogenic bacteria and diseases caused by the quorum sensing signal molecules dependent on AHLs is to treat crops by using the bacterial liquid of a Bacteroides nicotinovorans strain D-9 so as to prevent the infection of the pathogenic bacteria caused by the AHLs.

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

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

Experiments show that the Bacteroides nicotinovorans strain D-9 has obvious biological control effect on diseases caused by pathogenic bacteria of AHLs, namely, pectobacterium carotovorum subspecies (Pcc).

A degrading bacterial agent containing the Bacteroides nicotinovorans strain D-9 and/or bacterial liquid thereof and capable of degrading quorum sensing signal molecules AHLs and a biocontrol agent containing the bacterial strain D-9 and/or bacterial liquid thereof and pathogenic bacteria depending on AHLs for pathogenicity are also within the protection scope of the invention.

In order to achieve better effect of degrading the microbial quorum sensing signal molecules, the invention also provides a preparation method of the bacterial liquid of the bacterial strain D-9 in the nutrient-rich culture medium, which comprises the following steps: specifically, the strain D-9 is streaked on an LB solid 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 medium for pre-culture till logarithmic phase, and the strain D-9 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: 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 20 min. 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 finds that the Bacteroides nicotinovorans has quenching activity on a plurality of AHLs quorum sensing signal molecules, can rapidly and obviously degrade the AHLs quorum sensing signal molecules, 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 takes biological prevention and treatment instead of chemical prevention and treatment and blocks quorum sensing as a target without causing selective pressure.

Meanwhile, the invention obtains a bacteroides nicotinovorans (Ochrobactrum intermedium) D-9 strain through screening, the bacteroides nicotinovorans (Ochrobactrum intermedium) D-9 strain can normally grow in a culture medium with OHL with the concentration as high as 0.4mM as a unique carbon source, and a quorum sensing signal molecule OHL can be completely degraded within 32h, so that the bacteroides nicotinovorans (Ochrobactrum intermedium) D-9 strain has an obvious biodegradation effect. Meanwhile, the strain is used for other AHLs such as: OOHL and OdDHL also have better degradation effect.

According to the invention, because the strain D-9 has the characteristic of stably and efficiently degrading quorum-sensing AHLs signal molecules in plant pathogenic bacteria, the strain D-9 can be applied to the prevention and the treatment of AHLs mediated pathogenic plant pathogenic bacteria in natural environment, thereby not only reducing the abuse problem of pesticides, but also bringing new thinking, new approaches and new methods for the prevention and the treatment of plant diseases.

Drawings

FIG. 1 is a morphological diagram of the front and back sides of the bacterial strain D-9 of the present invention on LB medium.

FIG. 2 is a scanning electron micrograph of the strain D-9 of the present invention.

FIG. 3 is a phylogenetic tree analysis diagram of the strain D-9 of the present invention.

FIG. 4 is a graph showing the growth of strain D-9 of the present invention in various antibiotics.

Fig. 5 is a graph showing the results of the quenching activity of strain D-9 of the present invention on OHHL (e.coli is a negative control, B23 is a positive control).

FIG. 6 is an HPLC chart of OHL degradation by the strain D-9 of the present invention (line 1 is a chart of an uninoculated strain D-9, and lines 2 and 3 are High Performance Liquid Chromatography (HPLC) charts of 8h and 16h of OHL degradation by the strain D-9, respectively).

FIG. 7 is a graph showing growth curves and degradation curves of strain D-9 of the present invention using OHL as a sole carbon source.

FIG. 8 shows the disease onset of the strains D-9, E.coli, B23 of the present invention after being co-inoculated with Z3-3, respectively, to radish tubers 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 following examples are commercially available.

Example 1 acquisition and characterization of Bacteroides nicotinovorans strain D-9

1. Isolation and screening of Strain D-9

(1) Soil sample collection: soil samples collected from trees gardens and orchid greenhouses of southern China agricultural university as microbial sources

Soil sample separation from agriculture university in south China, Guangdong province, sampling, bagging, storing as microbial source and bringing back to laboratory for strain separation.

(2) Enrichment culture of the strain: basal salt (MSM) medium (formulation see Table 3) was prepared and dispensed into 100mL Erlenmeyer flasks at 50mL per flask. 5g of soil sample was weighed, aseptically added to an Erlenmeyer flask with soil sample (5g) and AHLs signal molecule (C6O3HSC,100mmol, 12.5. mu.L) and placed in a shaker at 30 ℃ at 200rpm for 7 days. After about one week, 1mL of the solution was taken out under aseptic conditions, the concentration of AHLs signal molecules was increased twice (i.e., 25. mu.L), and added to fresh MSM medium in a shaker at 30 ℃ at 200rpm for 7 days. By analogy, each soil sample is subjected to incremental enrichment culture for 8 times, bottles are replaced for 7 times respectively, and the concentration of AHLs signal molecules in a culture medium is increased continuously, so that the aim of enriching strains is achieved.

Wherein the formula of the basic salt (MSM) culture medium is as follows: 2g of ammonium sulfate; magnesium sulfate heptahydrate 0.2 g; 0.01g of calcium chloride dihydrate; 0.001g of ferrous sulfate heptahydrate; disodium hydrogen phosphate dodecahydrate 1.5 g; 1.5g of sodium dihydrogen phosphate; the pH was 6.5.

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

According to 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6Taking 1mL of fermentation liquor of the last MSM culture medium, sequentially diluting with sterile water, sucking 0.1mL of fermentation liquor with each concentration, uniformly coating the fermentation liquor on an LB solid plate by using a coating rod, and culturing for 24 hours in an incubator at 30 ℃. According to the observation, the solid culture medium with better single colony morphology and corresponding concentration is selected. Generally, a single colony is 10-4, 10^-5、10^-6The morphology is better in the concentration plate, and the single colony is relatively small and independently dispersed. Then, in the three concentrations of culture medium, a total of 35 different single colonies, respectively in 35 LB solid culture dishes in the form of a plate, marked, placed in 30 ℃ incubator for more than 24 hours. Colonies cultured for 24h above were scored as the first generation of purification. Picking out single colony from first generation culture medium, streaking and inoculating to new LB solid culture medium, and culturing at 30 deg.CAnd more than 24h, the product is regarded as the second generation of purification. By analogy, streaking and culturing were continued until the fifth purification generation was completed.

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

The deposited strains were removed at-80 ℃ in a refrigerator, the experimental strains were streaked onto LB solid plates, and the CF11 reporter strain was inoculated onto kanamycin plates, respectively. Placing the flat plate of the experimental strain in an incubator at 30 ℃ for culturing for 24 hours; plates of the CF11 reporter strain were placed in an incubator at 28 ℃ and incubated for 48 h. Individual colonies were picked separately with toothpicks on the plates and placed in tubes, each tube marked, and shaken overnight in a shaker at 30 ℃ and 200 rpm. Centrifuging the bacterial liquid (4000rpm for 5min), pouring out supernatant in an ultraclean workbench, adding 1mL of MSM liquid culture medium containing AHLs respectively, and setting a CK control tube; after 24h incubation, 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 of X-gal. 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: 2g of ammonium sulfate; magnesium sulfate heptahydrate 0.2 g; 0.01g of anhydrous calcium chloride; 0.005g of ferrous sulfate; 0.002g of manganese chloride; 10.5g of dipotassium phosphate; 4.5g of monopotassium phosphate; the pH was 6.5.

The principle related to the experiment is as follows: the AHLs signal molecules were able to diffuse along the agar strips of MM solid medium, with the diffusion distance being proportional to their concentration. When AHLs signal molecules exist on agar strips of the MM solid medium, a reporter strain CF11 corresponding to the strips starts to transcribe and express related genes of beta-galactosidase, and then the beta-galactosidase is released into the environment; at this point, X-gal in the medium contacts beta-galactosidase and is catalytically decomposed to produce 5-bromo-4-indigo. Wherein, X-gal is a colorless compound, and 5-bromo-4-indigo is a deep blue compound.

The blank contrast is LB liquid, without AHLs signal molecule degradation ability, corresponding to AHLs signal molecule concentration on agar strip high, diffusion distance long, beta-galactosidase normal expression, X-gal enzymolysis reaction, causing spot blue; in the screening bacteria, if the AHLs signal degradation molecule has strong ability, the spot can not be diffused in the environment, X-gal can not be decomposed, the spot is white, and if the AHLs signal molecule degradation ability is weak or the AHLs signal molecule degradation ability is not provided, the spot is blue. The more blue spots from both ends to the middle, the weaker the AHLs degrading ability and vice versa. The analysis experiment result shows that the strain marked as D-9 has stronger AHL quenching capacity and is to be further researched.

2. Identification and phylogenetic analysis of Strain D-9

(1) Colony morphology characteristics (shown in fig. 1): the bacterial colony is white, round, convex, opaque, neat in edge and smooth in surface after being cultured on a nutrient agar plate for 24 hours.

(2) Morphological characteristics of the thallus: as shown in FIG. 2, the cells are rod-shaped.

(3)16S rDNA sequence and phylogenetic analysis: the length of the 16S rDNA gene sequence of the strain D-9 is 1386bp, and the strain D-9 is compared with an NCBI database (http:// www.ncbi.nlm.nih.gov /), and the strain D-9 and Paenarthrobacter nicotinovorans are found to have good homology (> 99%), and the phylogenetic tree of the strain D-9 is shown in FIG. 3.

In conclusion, the 16S rDNA sequence and morphological characteristics of the strain D-9 are analyzed, so that the strain D-9 is identified as Bacteroides nicotinovorans (Paenarthrobacter nicotinovorans) and is preserved in Guangdong provincial microorganism culture collection 6-14 days 2019 under the accession number GDMCC No.60691 and the preservation address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5. .

Example 2 antibiotic susceptibility analysis of Strain D-9

1. In order to be able to better study the biocontrol potential of the strain D-9 obtained in example 1, we have conducted intensive studies on the biological properties of this strain. The sensitivity of strain D-9 to different antibiotics was investigated by the following method:

the strain D-9 is streaked and activated on a solid LB plate, a single colony is picked and inoculated to 1mL of liquidIn LB, the cells were cultured at 30 ℃ and 200rpm until the OD of the bacterial liquid₆₀₀The value is 1.0-1.5; each antibiotic was tested by setting 13 concentration gradients of 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL, 40. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, 200. mu.g/mL, 250. mu.g/mL, 300. mu.g/mL, 350. mu.g/mL, 400. mu.g/mL, respectively, and a blank control. 6 antibiotics kanamycin (kan), ampicillin (amp), gentamicin (gen), streptomycin (str), carbenicillin (carb), Tetracycline (TC), etc. were added at different concentrations to a centrifuge tube with 5mL of liquid LB, two for each concentration setting. Inoculating 1 μ L of bacterial solution into each tube, culturing at 30 deg.C and 200rpm in shaking table for 9 hr, and measuring OD of each tube of mixed system₆₀₀And (5) recording the values, and observing the growth conditions of the screened quenching bacteria in different antibiotic types and different antibiotic concentration gradients.

2. The experimental results show (as in fig. 4): the strain D-9 shows high-concentration sensitivity to gentamicin (gen) and carbenicillin (carb), and low-concentration resistance to 40 mg/mL; is highly sensitive to ampicillin (amp), kanamycin (kan), streptomycin (str) and Tetracycline (TC), and shows no resistance regardless of the concentration. This result is useful for reference in subsequent studies to select suitable antibiotics.

EXAMPLE 3 identification of quenching Activity of Strain D-9

1. Strain culture and sample collection: strain D-9 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 (Agrobacterium tumefaciens NT1) on the bottom. Agar to which the reaction mixture and the reporter strain have been spottedThe strips were placed in an incubator at 28 ℃ and incubated in the dark for 24h, and the experimental results were observed. Wherein the agar strips are obtained by cutting MM plates with the concentration of 40 mug/mL X-gal. 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 the strain D-9 has quenching activity on different AHLs.

2. In the experiment, Bacillus thuringiensis subsp. israelensis B23, which is known to quench a variety of AHLs, was used as a positive control, and Escherichia coli (Escherichia coli), which did not have AHLs-quenching activity, was used as a negative control. The test results show (as shown in fig. 5) that in the OHHL, OOHL and OdDHL test groups, the diffusion distances of the AHLs of the CK and the negative control e.coli are consistent, i.e. the AHLs content in the reaction mixture of the CK and the negative control e.coli is similar; the positive control B23 and the reporter strain on the agar strip of the experimental group D-9 did not turn blue, and the reaction mixture of the two did not contain AHLs, which were completely degraded by the strain D-9. The strain D-9 is proved to have quenching activity on an AHLs signal molecule OHL.

EXAMPLE 4 determination of the growth and degradation OHL relationship curves for Strain D-9

1. Inoculation: d-9 is streaked and activated from a refrigerator at the temperature of 80 ℃ below zero, after the culture is carried out for 24 hours at the temperature of 30 ℃, a single colony is picked to 8 to 10mL of liquid LB to be cultured by shaking to logarithmic phase (30 ℃,200 rpm), OD₆₀₀The value is about 1.0 to 1.5; a specific volume of the bacterial solution (V1/OD 600) was taken into a sterilized 2mL centrifuge tube, centrifuged at 10000rpm for 1min, and the supernatant was discarded. The precipitated cells were resuspended in a sterilized MSM medium, and then inoculated into 20mL of MSM medium, and AHLs stock solution (100 mmol/L) was added to give a final concentration of 0.4mmol/L of signal molecules in the medium system, followed by shaking culture at 30 ℃ and 200rpm, followed by sampling and detection at regular intervals. Sampling is set to be carried out once every 8 hours until the 48 th hour, and the time points are 8h, 16h, 24h, 32h, 40h and 48h, and the total number is 6 sampling points.

2. Sampling: in each medium, 7mL of each liquid was taken into a 15mL centrifuge tube, 1mL of which was used to measure OD₆₀₀Values (zeroing with ultrapure water) and recording; the remaining 6mL were centrifuged at 4000rpm for 5min, and 5mL of the supernatant was transferred to a glass graduated tube and allowed to stand for extraction.

3. Extraction and rotary evaporation: extracting the supernatant with equal amount of ethyl acetate, shaking in a separating funnel, standing for layering, recovering the lower layer liquid to the original glass scale test tube, waiting for secondary extraction, and filtering the upper layer liquid into a round-bottom flask through filter paper; after secondary extraction, removing lower-layer liquid, and filtering upper-layer liquid to the same round-bottom flask; fully evaporating the liquid in the round-bottom flask by a rotary evaporator, re-dissolving the attached substances in the round-bottom flask by using 2mL of acetonitrile, using 1mL of acetonitrile each time, repeating the steps, and completely transferring the substances into a clean test tube; and transferring the sample solution in the test tube to a chromatographic small bottle through a microporous filter membrane by using an injector, and sealing the bottle to be detected.

4. And (3) HPLC determination:

the respective parameters of the HPLC determination method for 3OC6HSL are as follows: HPLC: waters 2695; ② chromatographic column: KinetexEVOC18 reverse phase chromatography column (250 μm. times.4.6 mm. times.5 μm); flow velocity: 0.5 mL/min; column temperature: 30 ℃; mobile phase: acetonitrile: water 30: 70 (v: v); detection wavelength: 210 nm; sample introduction amount: 20 μ L.

5. The HPLC detection results are shown in FIG. 6 (wherein line 1 is a control chart of the non-inoculated strain D-9, lines 2 and 3 are degradation charts of the strain D-9 to OHL 8h and 16 h), and the degradation rates of the strain D-9 to OHL at 8h and 16h reach 91.8% and 99.8%, respectively. The growth curve and degradation profile of the corresponding strain D-9 using OHL as the sole carbon source are shown in FIG. 7.

As shown in FIG. 7, the growth rate of the strain is fastest, 40-48 h times, in 0-12 h. The degradation rate of the signal molecules AHLs (OHL) is the maximum within 0-12 h; at 12h, the signal molecules AHLs (OHL) were almost completely degraded. In the experiment, the degradation rates at 8h, 16h, 24h, 32h, 40h and 48h respectively reach: 91.8%, 99.8%, 99.9%, 100%, 100%, 100%. In conclusion, the growth of quenched D-9 and the degradation of the signal molecule are essentially positively correlated. The Bacteroides nicotinovorans D-9 has obvious and efficient degradation effect on AHLs signal molecules and has great application value for preventing and treating diseases mediating AHLs signal molecules to cause diseases.

EXAMPLE 5 biocontrol Effect of Strain D-9 on radish Soft rot

In this example, the biocontrol effect of strain D-9 on pathogenic bacteria that cause potato soft rot is studied, taking as an example the pathogenic bacterium, Pectiobacter carotovorus subsp.

In the inoculation test, the quenched D-9 bacteria, the Escherichia coli E.coli and the Bacillus thuringiensis B23 are all safe and non-pathogenic bacteria.

1. Bacillus thuringiensis B23, Escherichia coli E.coli, strain D-9, and carrot soft rot pectobacterium Z3-3 were streaked on 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-9 and pathogenic bacteria Z3-3 is adjusted to 1 x 10 by liquid LB⁷cfu/mL. Mixing the Z3-3 with B23, E.coli, D-9 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-9 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. As shown in figure 7, the lesion areas of the white radish tubers in the Z3-3+ LB and Z3-3+ E. coli experimental groups are obviously larger than those of the potatoes in the Z3-3+ B23 and Z3-3+ D-9 experimental groups, and the diseases are serious. Namely, the quenching bacterium D-9 and the pathogenic bacteria are inoculated together, and compared with the single inoculation of the pathogenic bacteria, no soft rot disease symptom appears.

Experimental results show that the strain D-9 has a certain biological control effect on the soft rot of white radish 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 (9)

1. A Bacteroides nicotinovorans (Paenarthrobacter nicotinovorans) strain D-9 is characterized in that the strain is deposited in Guangdong province microorganism culture collection center at 6 months and 14 days in 2019, wherein the deposit number is GDMCC No.60691, and the deposit address is as follows: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

2. Use of Bacteroides nicotinovorans strain D-9 according to claim 1 for the preparation of a product for degrading microbial quorum sensing signal molecules, which are N-Acyl Homoserine Lactones (AHLs).

3. Use of the Bacteroides nicotinovorans strain D-9 according to claim 1 for controlling plant diseases that are pathogenic mediated by or for producing a control agent for pathogenic bacteria that are pathogenic dependent on microbial quorum sensing signal molecules, such as N-Acyl Homoserine Lactones (AHLs).

4. The use according to claim 2 or 3, wherein the N-acyl homoserine lactone is OHL, OOHL or OdDHL.

5. A method for controlling pathogenic bacterial diseases caused by the dependence on microbial quorum sensing signal molecules, which are N-Acyl Homoserine Lactones (AHLs), comprising inoculating a crop with a bacterial solution of the Bacteroides nicotinovorans strain D-9 according to claim 1.

6. The method of claim 5, wherein the N-acyl homoserine lactone is OHL, OOHL or OdDHL.

7. The method of claim 5, wherein the pathogenic bacteria that are pathogenic in dependence on the microbial quorum sensing signal molecule include: a strain of Dickaurella (Dickeya sp.), a strain of Pectinobacterium (Pectiobacterium sp.), or a strain of Pseudomonas aeruginosa (Pseudomonas aeruginosa).

8. A quencher capable of quenching an quorum sensing signal molecule, which comprises the strain D-9 and/or a bacterial suspension thereof according to claim 1, wherein the quorum sensing signal molecule is N-Acyl Homoserine Lactones (AHLs).

9. A biocontrol agent for pathogenic bacteria which cause diseases by means of a microorganism quorum sensing signal molecule, which is N-Acyl Homoserine Lactones (AHLs), comprising the strain D-9 and/or a bacterial solution thereof according to claim 1.

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