KR100479884B1 - Microorganism Decomposing AHL, and Method of Using Thereof - Google Patents

Abstract

The present invention relates to a method for separating microorganisms degrading acylation homoserine lactone, which is a signal of density recognition from soil, and biologically controlling pathogenic microorganisms using the obtained microorganisms.

The new pathogenic microbial ecological approach will be able to effectively control pathogenic microorganisms while solving existing problems such as pesticide residue and antibiotic resistance.

Description

Acylated homoserine lactone-decomposing microorganisms and method of using the same {Microorganism Decomposing AHL, and Method of Using Thereof}

The present invention relates to acylated homoserine lactone (AHL) degrading microorganisms and methods of using the same. More specifically, the present invention relates to a method for separating microorganisms degrading acylated homoserine lactone, which is a signal of density recognition from soil, and biologically controlling pathogenic microorganisms using the obtained microorganisms.

Until now, chemical pesticides and antibiotics have been generally used to control bacterial diseases of crops. However, the use of the chemical pesticides and antibiotics have problems such as drug residues in the soil, killing of useful microorganisms, emergence of antibiotic resistant strains. As a way to overcome these problems, a new method is emerging that targets the density recognition signal transmission system of pathogenic microorganisms.

Just as insects release chemicals called pheromones to attract spouses or signal aggression, microbes also release chemicals associated with pathogenesis that have been found in Pseudomonas aeroginosa , a respiratory infection. These chemicals are called acylated homoserine lactones (AHLs), which primarily signal to produce molecules that cause the pathogenicity of flora and fauna pathogens, thereby causing disease.

Types of AHL include OHHL (N-β-oxo-hexanoyl-L-homoserine lactone), OOHL (N-β-oxo-octanoyl-L-homoserine lactone), and ODHL (N-β-oxo-decanoyl-L-homoserine lactone), HHL (N-hexanoyl-L-homoserine lactone), and BHL ( N- butanoyl-homoserine lactone).

In the case of Pseudomonas aeroginosa , which causes respiratory infections in patients with fibrocystic cysts, the concomitant signaling mechanism by acylated homoserine lactone has been elucidated, and development of new antibiotics through such disturbance This is in progress.

In addition, Korean Patent Application No. 10-1997-706614 discloses a method for controlling pathogenic microorganisms by using acylated homoserine lactone as a medium for signaling between microorganisms. However, the patent does not control pathogenic microorganisms through inhibition of acylated homoserine lactones themselves, but rather uses furanone and its derivatives that are structurally similar to acylated homoserine lactones to regulate proteins of acylated homoserine lactones. A method for controlling pathogenic microorganisms by competitively interfering with their binding. To date, no inhibition of acylated homoserine lactones has been studied.

An object of the present invention is to provide a microorganism containing an enzyme that decomposes acylated homoserine lactone, which is a density recognition signal substance between microorganisms.

In addition, an object of the present invention is to provide a method for fundamentally blocking the transmission of concomitant signal between microorganisms by decomposing concomitant signal material using the acylated homoserine lactone decomposing microorganism.

In order to achieve the above object, the present invention comprises the steps of (A) collecting a sample from soil, plants or animals; (B) inoculating the sample collected in a minimal liquid medium to which acylated homoserine lactones are added as the only carbon source to enrich the target microorganisms contained in the sample; And (C) smearing and enriching the enriched culture broth on an agar medium to obtain a single colony. Essentially, it provides a method for separating a strain having the ability to degrade acylated homoserine lactone.

That is, the ability to decompose acylated homoserine lactones by inoculating a sample taken from soil, plants or animals in a minimum medium containing any one or two or more substances of acylated homoserine lactones (AHL) as the sole carbon source. Enrich the desired microorganism with. Subsequently, the culture medium enriched in agar medium is plated and cultured to obtain colonies. If necessary, microorganisms are taken from a single colony, plated again on agar medium and cultured several times to obtain pure single colonies (strains).

As described in the following examples, it can be seen that the strain screened by the method according to the present invention can effectively degrade acylated homoserine lactones such as HHL and OHHL.

In addition, the present invention for achieving the above object, according to the method for separating the strain having the ability to decompose the above-described acylated homoserine lactone, aslobacter genus having an acylated homoserine lactone resolution ( Arthrobacter sp.) KH32 strain is provided. Thus, the isolated strain was able to decompose the acylated homoserine lactone which is a pathogenic derivative of the pathogenic microorganism (Autoinducer), it was confirmed that the actual pathogenesis is suppressed.

The present invention also provides a method and composition for inducing or enhancing disease resistance to pathogenic microorganisms of plants or animals using the aforementioned strains. More specifically, the above-mentioned strain Arthrobacter sp. KH32 may be sprayed and inoculated by spraying the surface of a plant or animal to induce or enhance resistance to pathogenic microorganisms. The present invention also provides a method for suppressing or treating a disease occurrence. The present invention also provides a bacterial or disease-inhibiting-treatment agent of a plant or animal comprising the above-mentioned strain as a main component, animal feed, fertilizer, pesticide, etc. containing the same. .

Hereinafter, the present invention will be described in more detail.

In order to obtain a microorganism that decomposes acylated homoserine lactone (AHL), which is a signal of density recognition, soil samples are inoculated into a minimal medium containing HHL (N-hexanoyl-L-homoserine lactone), a kind of AHL. And incubated. The culture sample was passaged three times and transferred to a nutrient broth agar plate and incubated for 48 hours to obtain a single colony.

In order to measure the AHL resolution of the obtained single colony-derived strains, a minimum of HHL and another AHL containing OHHL (N- [3-oxohexanoyl] -L-homoserine lactone) as the sole carbon source, respectively As a result of inoculation into the medium, cell growth by time and the residual amount of HHL and OHHL were measured, and it was confirmed that the isolated microorganisms degraded AHL.

Identification of isolated microorganisms confirmed that the genus Arthorbacter sp.

In order to determine the pathogenic microorganisms control ability of the microorganisms, it was confirmed that the application of potato stalk pathogenic strain Erwinia carotovora significantly reduced potato wort disease , and this isolate microorganisms control pathogenic microorganisms. It was.

Meanwhile, as a result of culturing fish pathogens Edwardsiella strains , Vibrio strains, and Aeromonas strains , their pathogens were BHL (N-butanoyl-). homoserine lactone) and HHL. Therefore, the fish disease can be controlled by utilizing the strain according to the present invention that can decompose the BHL and HHL.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Isolation and Identification of Acylated Homoserine Lactone Degrading Microorganisms

In order to screen the microorganisms that degrade the acylated homoserine lactone (AHL), which is a signal of density recognition, soil samples were collected from paddy fields located in Okcheon, Chungbuk, and enriched culture was carried out in a minimum medium.

Minimum medium (NaCl 1.5g / L, KCl 0.4g / L, MgCl 2 .6H 2 O 0.4g / L, CaCl 2 .2H 2 O 0.3g / L, KH 2 PO 4 0.2g / L, Na 2 SO 4 0.1g / L.MES 1g / L. NH4Cl 0.2g / L, HCl 100mM, FeSO4 · 7H2O 7.5mM, H3BO3 0.5mM, MnCl2 · 4H2O 0.5mM, CoCl2 · 6H2O 0.8mM, NiCl2 · 6H2O 0.1mM, CuCl2 · 2H2O 0.01mM, ZnSO4 · 7H2O 0.5Om 250 μg per ml of HHL was added as a sole carbon source to 2H 2 O 0.15 mM, Na 2 SeO 3 · 5H 2 O 0.02 mM, Na 2 WO 4 .2H 2 O, pH 6.0), and the inoculated medium was incubated at 30 ° C. for 72 hours.

The cultured samples were also subcultured three times by inoculating 5% (v / v) in a minimal medium containing 250 μg per ml of HHL. Culture conditions were the same as above. After the final subculture, the culture was streaking on a nutrient broth agar plate and incubated at 30 ° C. for 48 hours. Single colonies formed as a result of the culture were passaged three times in the same manner as described above to finally isolate a single species.

16S rDNA sequencing and Biolog analysis were performed to identify isolated AHL digested strains, and the morphological characteristics of the strains were examined.

(1) 16S rDNA Sequencing

16S rDNA sequences of the isolated strains were analyzed by conventional methods. The reaction conditions were as follows.

① primer: 9µl of distilled water, 0.4µL of 9F / 1542R (50X), 10µl of PCR master (2X), 0.5µl of template

② PCR conditions: 94 ℃ 5 minutes, 94 ℃ 30 seconds, 55 ℃ 45 seconds, 72 ℃ 2 minutes, 72 ℃ 2 minutes (30 cycles)

As a result of analyzing the partial sequence (SEQ ID NO: 1) and strain characteristics of the 16S rDNA amplified by PCR, it was confirmed that the isolated strain belongs to the genus Arthrobacter sp.

(2) Biolog analysis

The characteristics of the isolates isolated by conventional methods were investigated through Biolog analysis. Analysis conditions and results are shown in FIG. 1.

(3) other analysis

Gram staining revealed that the isolated strain was Gram positive. The isolated strain was also found to grow well at 30 ℃.

As a result of various analysis as described above, it was confirmed that the isolated strain belongs to the genus Arthrobacter sp. This was named KH32 and was deposited on October 20, 2001, with the accession number KCTC 10091BP to KCTC.

Example 2: Determination of AHL degradation of KH32 strain

In order to determine whether the KH32 strain obtained in Example 1 degrades AHL, the following experiment was performed.

OHHL and HHL, which are a kind of AHL, were used as test subjects. 5 mg of OHHL and HHL were dissolved per ml of ethyl acetate, and 0.1% (v / v) of glacial acetic acid was added to make a stock solution in a slightly acidic state and stored at -30 ° C. It was.

(1) HHL degradation measurement of KH32 strain

100 μl of HHL stock solution was removed in vacuo and then added to 2 ml of a minimum medium of the same composition as in Example 1. KH32 strain was inoculated into 2 ml of HHL-added medium and cultured at 30 ° C., and samples were collected by 100 μl at each time interval. The samples were analyzed to determine cell mass and residual HHL, respectively.

Cell mass was determined by measuring the OD value at 600nm using spectraMAX 190, HHL residual bioassay was performed as follows.

First, 100 μl of the sample taken at each time zone was extracted with 400 μl of ethyl acetate. Ethyl acetate was removed in vacuo and the sample was dissolved in at least 20 μl of medium. Chromobacterium vioraceum CV026 incubated in LB medium at 30 ° C. for 24 hours was overlaid on LB plates. After making a hole in the LB plate, extracted with ethyl acetate in the hole and put the 20μL sample dissolved in a minimal medium. After incubating the bioasaay plate overnight at 30 ° C., the diameter of the biolacein zone formed around the hole was obtained to measure the residual amount of HHL (FIG. 2). As shown in Figure 2 was confirmed that the isolated KH32 strain effectively degrades HHL within 24 hours. Since the KH32 strain was grown using AHL as the only carbon source, it was confirmed that the growth (OD value) of the strain and the reduction of AHL corresponded.

The actual degree of HHL degradation was calculated using the following correlation obtained by analyzing the relationship with violacein zone generated according to HHL concentration in CV026 plate.

Where X is the concentration of HHL (pmole), Y is the diameter of the zone (cm) and R-sqaure (reliability) is 0.98.

(2) OHHL degradation measurement of KH32 strain

Determination of OHHL degradation of KH32 strain was carried out in the same manner as in (1) except that OHHL was added instead of HHL. However, in order to increase the growth rate of the KH32 strain, an experiment in which 20 μl / ml of the vitamin solution was added to the minimum medium to which OHHL was added was also performed.

Bioassay was performed to measure the residual amount of OHHL in the culture medium.

50㎕ / ml salts (20X), 0.2% mannitol, 50㎕ / ml PO4 buffer (20X), in a minimal medium composed of 100㎍ / ml carbenicillin, the Agrobacterium at 30 ℃ cultivation for 24 hours Solarium tyumeo Pacific Enschede (Agrobacterium tumerfaciens ) Was overlaid on a minimal medium plate to which X-gal (40 µg / ml) was added. After making a hole in the bioassay plate, 20 μl of the sample diluted 60-fold in the hole was extracted with ethyl acetate and placed. After culturing the bioassay plate at 30 ° C. for 24 hours, the diameter of the blue zone generated as a result of β-galatosidase activity around the hole was determined to determine the amount of OHHL remaining (FIG. 3A).

After 19 hours in the minimal medium containing OHHL, the isolated strain had an OD of 0.21 and decomposed about 20 nmole of OHHL. After 72 hours, OHHL was decomposed 100 nmole with OD 0.3. In the medium to which the vitamin solution was further added, the OD reached 0.45 and the OHHL 125 nmole was decomposed after 19 hours (Table 1, FIG. 3B).

Incubation time OD value OHHL decomposition amount (nmol) 19 hours No vitamin 0.21 20 Vitamin addition 0.45 125 72 hours 0.3 100

In the pre-addition of X-gal plate agrobacterium by utilizing correlation below obtained by analyzing the relationship between the blue zone created according to the concentration of OHHL it was calculated actual degree of degradation HHL.

Where X is the concentration of OHHL (pmole), Y is the diameter of the zone (cm) and R-sqaure is 0.99.

Based on the above results, it can be seen that the isolated KH32 strain can decompose HHL and OHHL and grow by using the same. Thus, the strain KH32 according to the present invention is sprayed onto the surface of a plant or an animal. When spraying and inoculating by the method, it can be inferred that the strain can induce resistance to diseases or enhance resistance by decomposing signal material of pathogenic microorganisms. In addition, it will be possible to inhibit the bacterial disease development-treatment of a plant or animal comprising the strain according to the present invention, animal feed containing the same, fertilizers, pesticides and the like.

Example 3: Confirmation of AHL Degrading Enzyme Presence of KH32 Strain

In Example 2, it was confirmed that the KH32 strain grows by digesting AHL. In order to determine whether an enzyme that degrades acylated homoserine lactone exists in the KH32 strain, the following experiment was performed.

(1) Confirmation of HHL Degrading Enzyme of KH32 Strain

KH32 strains were inoculated in 5 ml of nutrient medium (Nutrient Broth, Merck) and then incubated at 30 ° C. for 24 hours. The culture was centrifuged at 12,000 rpm for 5 minutes and the supernatant and cells were separated separately. 500 μl of phosphate buffer (pH 6.5) was added to the obtained cells, the cells were treated with lysozyme, and sonicated to lyse the cells. The lysed cells were centrifuged at 12000 rpm for 5 minutes, and then only the supernatant was separated separately.

25 μl of the supernatant (corresponding to 250 μl of the cell) was added 175 μl of MES buffer (pH 6.5) containing HHL (HHL 10 μM) and reacted at 30 ° C. for 5 hours. In the same manner as in Example 2 (1), the diameter of the bioracein zone was measured using a bioassay plate in which the chromobacterium bioraceum CV026 was overlaid on the LB plate. As a result, it was confirmed that HHL of 1409 pmole was decomposed.

However, as a result of examining the HHL degradation activity in the supernatant of the cell culture medium in which the cells were not lysed, HHL was not degraded. Therefore, it was confirmed that HHL degrading enzyme is an enzyme present inside the cell rather than secreted out of the cell.

(2) Confirmation of OHHL Degrading Enzyme of KH32 Strains

The same method as in (1) above was used to confirm the presence of OHHL degrading enzyme of the KH32 strain. 50 µl of the obtained cell solution (corresponding to 500 µl of cells) was added to 150 µl of MES Buffer (pH 6.5) containing OHHL (OHHL 10 µM) and reacted at 30 ° C. for 5 hours. In the same manner as in Example 2 (2), the diameter of the blue zone was measured using a bioassay plate in which Agrobacterium tumenipians were overlaid on the LB plate. As a result, it was confirmed that OHHL of 1925 pmole was decomposed.

In the above results, the KH32 strain has an enzyme that degrades HHL and OHHL, which is a signal of density recognition, inside the cell, and it was confirmed that the enzyme degrades HHL and OHHL.

Example 4: Plant disease inhibition experiment using KH32 strain

(1) Confirmation of disease control ability of KH32 strain

We examined whether the KH32 strain, which degrades AHL, a density-recognition signal, actually has the ability to control plant diseases.

As plant pathogenic strains, Erwinia carotovora , which is known to be pathogenic due to AI (autoinducer), was used. Potatoes were used as crops to be applied. Experiments were carried out by treating the potato with varying concentrations of the pathogens Erwinia carotobora and KH32 strain.

The pathogen, Erwinia carotobora, was incubated at 30 ° C. for 16 hours in Nutrient Broth. 1% of the cultures were seeded in fresh Nutrient Broth and incubated at 30 ° C. for 5 hours.

The OD value of the Erwinia carotobora culture cultured as described above was adjusted to 1, and serially diluted from 10 ⁻¹ to 10 ⁻¹⁰ to inoculate potatoes at 20 μl. Potatoes were cut to 3 × 3 cm in length and used as they were, without any treatment.

The incidence of the disease was visually observed, and after 16 to 20 hours, a decayed and smeared form appeared, and the dark brown color of the potato was judged to have occurred.

The disease occurred when the OD value was 1 to 10 ^-4 , and the disease occurrence gradually decreased as the concentration of the pathogen decreased, and the disease did not occur at the concentration of the OD value of 10 ^-5 to 10 ^-10 (see FIG. 4). ).

KH32 strain was incubated for 16 hours at 30 ℃ in Nutrient Broth. 1% of the cultures were seeded in fresh Nutrient Broth and incubated at 30 ° C. for 5 hours.

The concentration of KH32 strain cultured as described above was adjusted to the OD value 0.1, 1 and 10.

Prepare the KH32 strain and Erwinia carotobora prepared as described above as shown in Table 2 strain mixture solution, and the surface of the potato cut 20 μl of a mixture of the strain mixture solution and 0.9% NaCl aqueous solution 1: 1: The inoculation was inoculated with a slight wound and confirmed the occurrence of the disease 24 hours later. At this time, the greater the number of +, the more severe the occurrence of the disease, which was determined by visual observation as described above (Fig. 4).

E NH32 0 10 One 10 ^-1 10 ^-1 +++ +++ +++ +++ 10 ^-2 ++ ++ ++ ++ 10 ^-3 + - + + 10 ^-4 - - - -

E: treatment concentration (OD) of Erwinia carotobora;

KH32: Treatment concentration (OD) of KH32 strain

+++ size of the disease about 2.5 cm; ++ about 1.5 cm in size of concomitant; + About 1 cm or less in size; -No involvement

As shown in Table 2 and Figure 4, when the concentration of the pathogen Erwinia carotobora 10 ^-4 , it was confirmed that the disease occurrence is suppressed regardless of the concentration of the KH32 strain. In addition, when the Erwinia carotobora concentration was 10 ^-3 , the disease occurrence was suppressed when the OD of the treated KH32 strain was 10, but the disease occurred at other concentrations. In the above results, it was confirmed that the KH32 strain inhibits the pathogenesis of pathogens.

(2) Confirmation of disease suppression mechanism of KH32 strain

It was confirmed whether the strain KH32 according to the present invention kills the pathogenic strain to inhibit the disease, decomposes the AI (autoinducer) material such as AHL to suppress the disease, and whether the pathogenic strain is alive at the site of inoculation.

First, the culture medium (E + KH group) was cut so that the OD of the pathogens Erwinia carotobora (OD = 0.001) cultures (E group), the Erwinia carotoboro and KH32 strains obtained in the previous (1) became 0.001 and 0.1, respectively. The surface of the prepared potatoes was inoculated. Inoculation method was the same as above (1).

After 24 hours, the E group developed, and the E + KH group confirmed that the disease was suppressed. Subsequently, 50 μl of 100 μM of OHHL was treated as AI on the surface of the E + KH group potatoes in which the disease was suppressed. After 24 hours, the disease was similar to that of the E group in the E + KH group potato, which was initially suppressed (FIG. 5).

As can be seen in the figure, it was confirmed that pathogens were also alive at the site where the disease was suppressed by the KH32 strain, that is, the KH32 strain did not directly kill the pathogen but indirectly prevented the pathogenesis by decomposing AI.

Presumed Example 5: Fish disease pathogen control analysis by KH32 strain

In Example 4, it was examined whether the KH32 strain which is effective in controlling plant diseases is also effective in fish pathogenic microorganisms.

A variety of substances produced from fish strains to confirm whether the signal substance produced by the bacteria showing the fish pathogenicity is actually degraded by the signal substance decomposing strain KH32 isolated in the present invention, and whether the KH32 has the possibility of controlling fish disease. Signals were investigated.

Edwardsella sp. And Vibriosi s sp., The causative agents of Edwardsieuosis from dead flounders, and Aeromonas sp., Which cause redness on the skin and fins, Was isolated from the kidneys of dead sweetfish.

Edema crushed dead kidney fish was inoculated in SS (Salmonella Shigella agar) selective medium and cultured at 30 ° C., colony of which the center of colony was turned black was selected to isolate Edward Siela species. Also, the dead fillet kidney was inoculated in TCBS (Thiosulfate citrate salts agar) and incubated at 25 ° C. to select yellow colonies to isolate vibrio species.

In the case of aeromonas species, the kidney crushed dead fish was inoculated in TSA (Tryptic Soy Agar), and then cultured at 25 ° C. for 48 hours to obtain a colony showing a brown water-soluble pigment.

Various fish disease pathogens isolated through the above method were incubated for 24 hours at 25 ° C. in LB medium, and then the culture solution was extracted with ethyl acetate solvent to perform TLC (thin layer chromatograph). 60% methanol was used as the developing solvent. As a result of examining the signal material developed on the TLC, the main signal material of the isolated pathogens was identified as BHL ( N -butanoyl-homoserine lactone) and HHL ( N -hexanoyl-homoserine lactone) (Fig. 6).

Since all of the signal substances can be decomposed by the isolate KH32, it will be able to control the disease of the fish by using the strain KH32 according to the present invention.

According to the present invention, microorganisms capable of degrading acylated homoserine lactone, which is a signal of density recognition of pathogens, can be separated purely, and biologically controlling pathogenic microorganisms using the strains separated by this method.

The new ecological approach to controlling pathogenic microorganisms is useful as an effective and environmentally friendly pathogenic microorganism control method while solving the problems of pesticide residue and antibiotic resistance, which are problems of conventional biochemical pathogenic microorganism control.

1 is a table showing the Biolog analysis conditions and results of isolated KH32 strain.

Figure 2 is a graph showing the growth and degradation in the minimal medium added HHL of isolated KH32 strain.

3A is a bioassay plate photograph showing degradation of OHHL by isolated KH32 strains.

Figure 3B is a graph showing growth and degradation in the minimal medium with OHHL added KH32 strain isolated.

4 is a pathogenic strain of isolated KH32 strain Erwinia Carotoborah Photo of experimental results showing control of potato blight.

Figure 5 is a photograph of the experimental results that can infer the disease control mechanism of the isolated KH32 strain.

Figure 6 is a TLC result photograph showing the type of AI secreted by fish disease pathogens.

KU, Bon-Tak; InBioNET Corp. <120> Microorganism Decomposing AHL, and Method of Using Thereof <130> P1101-006 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 256 <212> DNA <213> Arthrobacter sp. <400> 1 ccccccccnc ttttannngn ggataatatc gnctggtcgg acnnanccnt tnnnntatgg 60 cangtncgaa cgaatgcntc cccagcttgc tgggggatta ntggcgaacg ggtgagtaac 120 acgtgagtaa cctgcccttg actctgggat aagcctggga aactgggtct aataccggat 180 atgactcctc atcgcatggt ggggggtgga aagcttttgt ggtaattgga tggantnnaa 240 gactatcatc ttgttt 256

Claims (7)

delete A strain of Arthrobacter sp. KH32 (KCTC 10091BP) that has the ability to degrade acylated homoserine lactones, a microbial density signal. A method for inhibiting-treating bacterial disease occurrence by spreading the strain according to claim 2 on the surface of an animal except a plant or a human . delete delete delete delete