KR20120055801A - Bacteriophage having specific lysis activity for bacterial wilt pathogen in plant - Google Patents

Abstract

PURPOSE: A bacteriophage isolated from soil is provided to ensure specific lysis activity toward Ralstonia solanacearum and to develop an agent for preventing bacterial wilt in various plants. CONSTITUTION: Bacteriophage PE226(deposit number: KACC 97007P) has a lysis activity specific to Ralstonia solanacearum. The bacteriophage has a genome having a base of sequence number 1. The bacteriophage is active at 25°C-60°C and a pH of 4-11. A composition for preventing plant diseases caused by the Ralstonia solanacearum contains the bacteriophage as an active ingredient. The plant diseases include plant bacterial wilt. A method for preventing plant diseases comprises a step of applying the composition onto plants.

Description

Bacteriophage having specific lysis activity for bacterial wilt pathogen in plant}

The present invention relates to a bacteriophage having a specific lytic activity against plant foot blight bacteria, more particularly bacteriophage (bacteriophage) having a specific lytic activity against Ralstonia solanacearum , the bacteriophage Control of ralstonia solana serum-induced plant diseases comprising the step of treating the plant with the composition for the control of ralstonia solanaserum-induced plant diseases comprising the active ingredient and the composition comprising the bacteriophage as an active ingredient It is about a method.

Ralstonia Solana Serum solanacearum ) is a phytopathogenic Gram-negative bacterium that causes foot blight by adding more than 50 and 200 species of plants, and secretes extracellular polysaccharides (EPS) and hydrolases that can degrade plant cell walls as pathogenic factors. (Schell, Annu . Rev. Phytopathol. 38: 263-292, 2000). In particular, infection through the roots of plants and invasion of the water pipes of the plant produces a large amount of EPS and blocks the water pipes of the host plants, which is a major cause of foot blight in branches and crops (Saile et al., Phytopathology 87: 1264-). 1271, 1997). In soil environments, Ralstonia solanaserum strains are easily spread in irrigation water and are known to survive long in the soil. But Lal Stony Oh Solana, foot blight of Solanaceae crops by the serum is not high, the various control methods efficiency-resistant crops almost no as well as temperate, subtropical and tropical regions worldwide, is a serious plant disease (Hayward, Annu. Rev . Phytopathol 29:. 65-87, 1991 ).

Antibiotics have been used for more than half a century to control various pathogenic bacteria, leading to the emergence of a large number of antibiotic resistant bacteria, and the emergence of resistant strains compared to the development of new antibiotics, a new bactericidal method to replace antibiotics. This is the time needed. Bacteriophage is a virus that specifically infects bacteria to lyse or tolerate bacteria, and infects only prokaryotic bacteria as compared to viruses of eukaryotic animals or plants. Therefore, bacteriophages are being actively researched as an alternative way to control bacteria instead of antibiotics. Recently, attempts to control food contaminant Listeria monocytogenes by bacteriophages, rather than antibiotics, have been approved by the US Food and Drug Administration (Federal Register, vol 71. No. 160 page 47729-47731). , 2006). In the case of plant bacterial diseases, attempts are being made to use bacteriophage instead of antibiotics, because bacteriophage has a shorter development time and a lower cost than antibiotics (Jones et al., Annu. Rev.). Phytopathol. 45: 245-262, 2007). Recently, Yamada et al. ( Microbiology 153: 2630-2639, 2007) have isolated a new class of bacteriophages that specifically infect Ralstonia solanaserum, which are used to detect bacteria, study pathogenicity, and control crop bacterial diseases. Can be used. Recent studies have shown that the total number of viruses present in the global ecosystem is more than a few times larger than the total number of bacteria, and the development of technology to control bacteria using them has many possibilities (Fierer et al., Appl . environ Microbiol 73:.. 7059-7066, 2007).

Accordingly, the present inventors have selected a bacteriophage capable of lysing various strains of Ralstonia solanaserum to isolate bacteriophages lysing Ralstonia solanaserum strains from the soil. The selected bacteriophages, in comparison with the conventionally known bacteriophages, differ from the typical linear virus in the form of a flexible linear virus, which is morphologically distinct, and has a large number of specific genes as a result of analyzing the genome sequence. It was confirmed. In addition, the bactericidal activity against other bacteria was confirmed, and the plant pathogenicity showed widespread lytic activity against various Ralstonia solanaserum strains, and the present invention was completed by confirming the control effect of plant foot blight disease such as tomato.

Korean Patent No. 10-0943041 discloses bacteriophages having killing ability against E. coli or Salmonella, and Korean Patent No. 10-0781669 discloses bacteriophages having Staphylococcus aureus specific killing ability.

The present invention is derived from the above requirements, and the present inventors have confirmed that the bacteriophage isolated from the soil has a specific lytic activity against Ralstonia solanacearum , a causative agent of plant foot blight. The invention was completed.

In order to solve the above problems, the present invention provides a bacteriophage having a specific lytic activity against Ralstonia solanacearum .

The present invention also provides a composition for controlling Ralstonia solana serum-inducing plant disease, comprising the bacteriophage as an active ingredient.

The present invention also provides a method for controlling Ralstonia solana serum-induced plant disease, comprising treating the plant with a composition comprising the bacteriophage as an active ingredient.

Since bacteriophage PE226 of the present invention has specific lytic activity against Ralstonia solanacearum , it is expected that the bacteriophage PE226 may be useful for controlling plants with green blight.

Figure 1 shows the formation of lysate for Ralstonia solanaserum after pure separation of bacteriophage PE226.
Figure 2 shows the form of the bacteriophage PE226 observed using an electron microscope. Bar = 100 nm.
3 shows DNA isolated from bacteriophage PE226 and DNA digested by treating it with RNase, EcoRI and Nuclease S1. M: marker DNA (lambda DNA digested with restriction enzyme HindIII), lane 1: PE226 genomic DNA, lane 2: PE226 genomic DNA (RNase treatment), lane 3: PE226 genomic DNA (Nuclease S1 treatment), lane 4: PE226 genome DNA (EcoRI treatment).
Figure 4 shows the total DNA sequence of the bacteriophage PE226.
Figure 5 shows the results of analyzing the gene sequence and similarity of the bacteriophage PE226 gene with other bacteriophage genes. Each number is an ORF (open reading frame) number. The numbers under p12J and φRSS1 are ORF numbers similar to the ORFs of PE225, with the percentage indicating the identity of the gene sequence for each ORF of PE226. Below is a table of the results.
6 analyzes the stability of bacteriophage PE226 at various temperatures.
7 analyzes the stability of bacteriophage PE226 at various pHs.
FIG. 8 shows the results of testing foot blight control effect using bacteriophage PE226 in tomato seedlings. FIG. The x-axis represents the date after treatment of the pathogen and phage, and the y-axis represents the onset index. The standard deviation for the mean of each 6 replicates is shown in bars.
9 is a photograph showing the control effect of foot blight disease using bacteriophage PE226 in tomato seedlings. (A) Bacteriophage PE226 alone treatment, (B) Pathogen SL341 alone treatment, (C) Pathogen and PE226 mixed treatment (PE226's foot blight control effect).

In order to achieve the object of the present invention, the present invention provides a bacteriophage having specific lytic activity against Ralstonia solanacearum .

Preferably, the bacteriophage is bacteriophage PE226 (Accession Number: KACC 97007P).

Preferably, the bacteriophage may have a genome consisting of the nucleotide sequence of SEQ ID NO: 1. In addition, the present invention is another bacteriophage having a genome having high identity (preferably at least 70%, more preferably at least 80%, most preferably at least 90%) with the base sequence of SEQ ID NO: 1 It may include.

Preferably, the bacteriophage may have activity at 25 ° C. to 60 ° C., but is not limited thereto. Preferably, the bacteriophage may have activity at pH 4 to 11, but is not limited thereto.

The bacteriophage PE226 was selected from bacteriophages with lytic activity against Ralstonia solanaserum among the bacteriophages isolated from the soil. The bacteriophage PE226 was deposited on November 10, 2010 at the National Institute of Agricultural Science, Agricultural Genetic Resources Center (Accession Number: KACC 97007P).

In addition, the present invention includes the bacteriophage as an active ingredient, Ralstonia solanaserum ( Ralstonia solanacearum ) provides a composition for controlling the induced plant disease. Preferably, the Ralstonia solanaserum-induced plant disease may be plant foot blight, but is not limited thereto. Preferably, the plant may be a dicotyledonous plant, but is not limited thereto.

The dicotyledonous plants include Solanaceae, Amphiaceae, Diapensiaceae, Cacthraceae, Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae, and Primaceae , Ebenaceae, Styracaceae, Stink bug, Symplocaceae, Ash (Olanaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae Apocynaceae, Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Lamiaceae, Labiatae, Scrophulariaceae , Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae , Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Landaceae, Phytolaccaceae, Nyctaginaceae, Pomegranate, Azizaceae (Portulacaceae), Caryophyllaceae, Chinopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, Ranunculus eae), Berberidaceae, Lardizabalaceae, Bird breeze (Mentaceae, Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Papaveraceae ), Capparidaceae, Cruciferaceae (Mustaceae, Cruciferae), Planeaceae (Plataceae, Platanaceae), Verruaceae, Hamamelidaceae, Pheasant (Snaphaceae, Crassulaceae), Panaxaceae (Saxifragaceae) Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae Euphorbiaceae), star moss (Callitrichaceae), Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Mapleaceae Hippocastanaceae, Sabiaceae, Balsam, Balsaminaceae, Aquifoliaceae, Nova, Celastraceae, Staphyleaceae, Buxaceae, Empetraceae, Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Eraeagnaceae (Flacourtiaceae), Violaceae, Passifloraceae, Tamaricanaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Buddha (Lythraceae), Pomegranate Punica ceae), Onnagraceae, Haloragaceae, Bataceae (Alangiaceae), Dogwood (Hornaceae, Cornaceae), Arboraceae (Agaraceae) or Umbelliferae (Apiaceae) It may be, but is not limited thereto. Preferably, the dicotyledonous plant may be Solanaceae, most preferably tomato, potato or pepper, but is not limited thereto.

Compositions for controlling Ralstonia solanaserum-induced plant diseases according to the invention are for example in the form of direct sprayable solutions, powders and suspensions, highly concentrated aqueous and oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, It may be prepared from dust, scattering material or granules, but is not limited thereto. In addition, the composition for controlling the Ralstonia solana serum induced plant disease may be used by spraying, spraying, spraying, spraying or pouring. The form of use depends on the intended purpose and in all cases the distribution of the composition according to the invention should be as fine and uniform as possible.

The composition for controlling the Ralstonia solanaserum-inducing plant disease of the present invention can be formulated in various forms. Such formulations may be prepared, for example, by adding solvents and / or carriers. Often, inert additives and surface-active substances such as emulsifiers or dispersants are mixed in the formulation. Suitable surface-active substances include aromatic sulfonic acids (eg lignosulfonic acid, phenol-sulfonic acid, naphthalene- and dibutylnaphthalenesulfonic acid), fatty acids, alkyl- and alkylarylsulfonates, alkyl lauryl ethers, alkalis of fatty alcohol sulfates. Of metals, alkaline earth metals, ammonium salts, sulfated hexa-, hepta- and octadecanols, salts of fatty alcohol glycol ethers, sulfonate naphthalenes and derivatives thereof, condensates of formaldehyde, naphthalene or naphthalenesulfonic acids, phenols and formaldehydes Condensates, polyoxyethyleneoctyl phenol ethers, ethoxylated isooctyl-, octyl- or nonyl-phenols, alkylphenyl or tributylphenyl polyglycol ethers, alkylarylpolyether alcohols, isotridecyl alcohols, fatty alcohol / ethylene oxide condensation Water, ethoxylated castor oil, polyoxyethylene alkyl ether or polyoxypropylene, lauryl alcohol All polyglycol ether acetate, sorbitol ester, lignin-sulfite waste liquor or methylcellulose. Suitable solid carrier materials are in principle all porous and agriculturally acceptable carriers, for example mineral earth (such as silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, bowls, loess, clays, Dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers (such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), vegetable products (such as grain flour, bark flour, wood meal) And nut shell powder) or cellulose powder, but is not limited thereto. In addition, the said solid carrier can also be used 1 type or in mixture of 2 or more types.

In addition, the composition for controlling the Ralstonia solanaserum-induced plant disease may be mixed with a diffusing agent, a penetrant or a surfactant to enhance crop absorption and effect.

In addition, the composition for controlling Ralstonia solanaserum-induced plant diseases of the present invention may be formulated into a pesticide formulation further comprising a pesticide that has a control effect against plant infectious diseases.

In addition, the present invention Ralstonia Solanaserum comprising the step of treating the plant with a composition comprising the bacteriophage as an active ingredient ( Ralstonia) solanacearum ) to provide a method of controlling the induced plant diseases. Preferably, the Ralstonia solanaserum-induced plant disease may be plant foot blight, but is not limited thereto. Preferably the plant may be, but is not limited to, a dicotyledonous plant. The plant is as described above.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: Separation of Bacteriophage

The soil samples used in this study were obtained from tomato, pepper and tobacco growing soils in Cheongsong, Nutrition, and Gangseo-gu, Busan. The collected soil was filtered through a sieve 1 mm in diameter, and 50 g of soil was added to the sterilized water to contain a total of 40% water. 2 ml of Ralstonia solanaserum strain was added to each soil to 10 ⁸ CFU / ml, and 0.5 ml of 1% glucose was added and stored for 48 hours. 10 ml of phosphate buffered saline (phosphate buffered saline, PBS, pH 7.0) was added per 5 g of the enriched soil, and suspended at 150 rpm at room temperature for 10 minutes. The soil suspension was centrifuged at 10,000 rpm for 20 minutes at 4 ° C. and the supernatant was collected and filtered through a 0.2 μm diameter membrane. In order to concentrate the bacteriophage particles of the recovered supernatant, 2 ml of the supernatant was mixed with 1 ml of Ralstonia solanaserum overnight culture and maintained at 30 ° C. for 24 hours. Thereafter, the bacteria were lysed by treatment with 3 ml of 10% chloroform, and centrifuged at 10,000 rpm to remove bacteria residues.

The presence of bacteriophages was confirmed by observing the formation of plaques by performing a soft agar stratification in CPG medium. This was mixed with 1 ml of bacterial culture solution and 0.5 ml of bacteriophage solution, mixed with CPG medium containing 0.7% agar, layered on normal CPG solid medium, and culturing at 30 ° C to observe formation of lysate plaques. Single lysates were selected from each sample, and again single lysates were selected by the semi-solid agar stratification method. The procedure was repeated three times in succession to finally separate the bacteriophages that form a pure single lysate.

Figure 1 shows the formation of lysate plaques after culturing mixed bacteriophage pure host bacteria. Selected bacteriophages were grown in the corresponding host bacteria and then suspended in SM buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 10 mM MgSO ₄ and 0.01% gelatin) and stored at 4 ° C.

Example  2: wide range Host  infection Ralstonia  Specific Bacteriophage Selection

The host range of the obtained bacteriophage was determined by two methods for eight Ralstonia solana serum strains. That is, after inoculating the bacteriophage solution into the host bacteria and culturing, the method of observing the formation of lysate plaques in the solid medium and the method of dropping the bacteriophage solution into the medium on which the host bacteria were smeared were used. The strains (race, biotype) of nine Ralstonia solana serum strains used are shown in Table 1 below.

Ralstonia solanaserum strains used to determine host range Strain name Separation host Race Biotype SL312 tomato One 3 SL341 tomato One 4 SL2268 potato 2 2 SL2029 potato 2 2 SL2064 potato 2 2 SL2312 potato 2 2 SL2313 potato 2 2 SL3531 pepper One 4 GMI 1000 tomato One 3

The concentration of each bacteriophage used for determination of the host range was adjusted to 10 ⁵ PFU / ml by a multi-step dilution method. The bacteriophage solution was incubated for 10 hours at 30 ° C. by titrating 10 μl of each host bacterium onto the dehydrated medium. And lysate plaque formation was observed. In addition, bacteriophages of the same concentration were mixed with 10 ⁷ CFU of bacterial suspension, mixed with 3 ml of top agar, stratified and incubated at 30 ° C. for 24 hours to observe the formation of lytic plaques. In both cases, it was confirmed that the host bacteria could be lysed only when a clear lysate plaque was formed. As a result, among 15 purely isolated bacteriophages, bacteriophage PE226 that can infect all the host was selected. In other words, PE226 was determined to be a broad host lytic bacteriophage capable of lysing most pathogens regardless of race or biotype of Ralstonia solanaserum strain. However, PE226 was used in the same way as Pseudomonas fluorescens , Burkholderia pyrrocina ), Escherichia coli ) showed no lytic activity at all. Therefore, it was confirmed that the PE226 selected in the present invention is a bacteriophage that is specific to Ralstonia solanaserum and capable of lysing a wide range of host bacteria.

Example  3: bacteriophage PE226 Purification and Morphology Analysis

In order to purify the bacteriophage PE226 selected in the present invention, a high concentration of phage solution was first inoculated into 10 ⁸ CFU / ml of Ralstonia solanaserum SL2268 strain in algebraic incubator and incubated in 4 ml CPG liquid medium for 24 hours. . After removing the bacteria from the culture and partially purified by eluting the bacteriophage eluted, a total of 4 ml inoculated in 1000 ml of host bacterial suspension culture grown to the algebraic period using the SL2268 strain and further incubated for 48 hours at 30 ℃ It was. The mixture was treated with a small amount of chloroform, mixed, and centrifuged at 5,000 rpm for 15 minutes to recover the supernatant. The recovered supernatant was repeatedly centrifuged in the same manner to remove host bacteria, and NaCl was added to the total supernatant so that the final 1 M was stirred for 1 hour while stirring the supernatant until the NaCl was completely dissolved. After centrifugation at 11,000 rpm for 15 minutes at 4 ℃, the supernatant was recovered to add PEG 8000 to the final 10% and maintained overnight at 4 ℃, centrifuged for 30 minutes at 4 ℃ 11,000 rpm for bacteriophage concentration. . The supernatant was then removed and the precipitate resuspended in 500 μl SM buffer.

The morphology of the purified bacteriophage by PEG precipitation was measured by dropping 5 μl of the purified bacteriophage solution onto the Formvar-carbon-coated copper grid and using filter paper to remove excess moisture. After removal, 3 μl of 2% sodium phosphotungstate (pH 7.4) was added and stained. After about 2 to 5 minutes, water was removed and the copper grid was dried in air for 10 minutes and used for electron microscope observation. The electron microscope was observed at 200 kV using JEM-2010 (JEOL, Japan) as a transmission electron microscope. . The observed PE226 was a long filamentous bacteriophage similar to that of the Inoviridae virus as shown in FIG. 2. The average length of the bacteriophage PE226 is about 1,050 ± 200 nm and the width is about 7 ± 1.5 nm. In addition, it appeared as a filamentous bacteriophage that was not straight but semicircular or flexible.

Example  4: bacteriophage PE226 Genome separation analysis

DNA isolation of bacteriophage PE226 was isolated using purely isolated bacteriophage solution according to the methods of Sambrook and Russell (Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbor, NY, 2001). Encapsulated protein was removed using proteinase K, and total nucleic acid was separated from 10 ml of pure phage solution using phenol-chloroform-isoamyl alcohol.

In order to confirm the characteristics of the isolated bacteriophage genome, restriction enzymes such as RNase and Eco RI were treated, and no change was observed when the separated genome was compared with the untreated genome on agarose electrophoresis. Thus, the genome of PE226 was identified as DNA and was not sensitive to restriction enzyme treatment, thus testing the possibility that the DNA of PE226 was a single helix. Nuclease S1, which selectively degrades single-stranded DNA, was treated with DNA isolated from PE226, confirming that DNA was completely separated. Thus, the DNA of PE226 was confirmed to be a single strand. Figure 3 shows the isolated DNA of PE226 and the DNA digested as a result of treatment with Nuclease S1.

In order to determine the nucleotide sequence of a single strand of DNA, a double strand of DNA must first be converted. In the present invention, multiple displacement amplification (MDA) was performed to recover the double strand of DNA. The MDA reaction was carried out according to the method using the TempliPhi 100 amplification kit (GE Healthcare, UK) to recover the double-stranded DNA, the MDA reaction product, which was cut with various restriction enzymes, cloned and inserted into pUC119 cut with the same restriction enzyme. The base sequence of the DNA was determined. In order to eliminate the error of bacteriophage DNA amplification by the MDA method, the DNA base sequence of PE226 shown in FIG. 4 was again determined by primer walking using the DNA of PE226 as an original. FIG. 4 shows the nucleotide sequence of PE226, which is partially similar to bacteriophage φRSS1 infecting Ralstonia solanaserum or p12J infecting other Ralstonian species, but is a kind of two different bacteriophages φRSS1. Alternatively, the nucleotide sequence of the p12J genome was mixed. In terms of overall gene configuration and arrangement, PE226 showed a significant difference from φRSS1 or p12J, and the results show the similarity of gene arrangement in FIG. 5. A total of 5,475 pairs of DNA sequences and about 9 ORFs (open reading frames) were identified from PE226. This is considerably less than the base sequence of 7,118 pairs and 6,662 pairs of DNAs of p12J and φRSS1. The predicted function of each predicted ORF is shown in FIG. 5 together, but the function of many genes is unclear.

Example  5: bacteriophage PE226 Stability

The stability of the selected PE226 at various temperatures, pH and organic solvent conditions was tested. In order to investigate the stability at various temperatures, the pure PE226 solution suspended in SM buffer was maintained at various temperatures for 1 hour, and diluted in 10 steps to perform a 0.7% top agar stratification method. 10 μl of the diluent of each step was mixed in the tower agar, mixed with Ralstonia solanaserum strain SL2268, and cultured in a solid medium to count the lysate plaques. As a result, PE226 was stable from 25 ℃ to 60 ℃, 50% of the total bacteriophage was maintained at 65 ℃. The results are shown in FIG.

The pH stability of PE226 was determined by various pH solutions (sodium citrate buffer (pH 3.0, pH 4.0 and pH 5.0); sodium phosphate buffer (pH 6.0 and pH 7.0); Tris-HCl (pH 8.0, pH 9.0, pH 10.0 and pH 11.0). )) Were prepared by 2 M each and investigated. 100 μl of the pH solution and the same amount of 1.0 × 10 ¹¹ PFU / ml bacteriophage solution were mixed so that the concentration of each pH solution was 1 M, followed by standing at room temperature for 1 hour. This was diluted in 10 steps and 0.7% tower agar stratification was performed. 10 μl of the diluent of each step was mixed in the tower agar, mixed with Ralstonia solanaserum strain SL2268, and cultured in a solid medium to count the lysate plaques. As a result, it was found that PE226 had a 25% decrease in activity at pH 3 but was very stable without losing activity from pH 4 to pH 11. The results are shown in FIG.

Example  6: bacteriophage PE226 Using Plant disease  Control effect

In order to test whether the selected bacteriophage PE226 can effectively suppress crop foot blight, tomato plant seedlings were used to investigate the disease. Tomato seeds used in the experiment were surface sterilized in 0.1% NaOCl solution for 3 minutes and germinated by washing at least 5 times with sterile water. The tomato seedlings grown for three weeks were grown in pots containing non-concrete soils and treated with pathogens SL341 and PE226 to test their inhibitory effects. The pathogen Ralstonia solanaserum SL341 strain was incubated overnight at 30 ° C. with CPG liquid medium, and only bacterial cells were recovered and suspended in sterile water to adjust the absorbance to 0.5 at 600 nm to prepare an inoculum. Bacteriophage PE226 was used at a concentration of 10 ⁸ PFU / ml. The pathogen SL341 strain and bacteriophage PE226 were mixed in a 1: 1 ratio and fertilized in a pot where tomato plants were grown at a concentration of 10 ⁷ CFU / ml of pathogen per gram of soil. The control group was irrigated with only SL341 strain or with PE226 alone, and the untreated control was treated with 1: 1 mixing of sterile water and SM buffer. The plant contained 6 replicates per treatment. All treated plants were repeatedly maintained at 32 ° C, 14 hours of light conditions, and 25 ° C, 10 hours of cancer conditions, and daily disease occurrence was observed by observing the degree of plant withering, which is the standard quantitative method of foot blight disease. Quantitation was by the index (disease index). The incidence index 0 shows no wilting at all, the incidence index 1 withered at 25%, the incidence index 2 at 50%, the incidence index 3 at 75%, and the incidence index 4 at all plants withered. Corresponding. 8 is a graph showing the onset situation and FIG. 9 is a photograph showing the onset situation.

Institute of Agricultural Biotechnology KACC97007 20101110

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Bacteriophage having specific lysis activity for bacterial wilt          pathogen in plant <130> PN10241 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 5475 <212> DNA <213> Bacteriophage PE226 <400> 1 atggaaatga tcgcgcgcgt caccattcgg ggcgcaaaga cgtgggtagg cgtgatggaa 60 ggcaagcaac tcgataccgg cacgatctac gttgatgtcg agctgcgtgg cgaggactcg 120 aagggcacct gcacgcaggc gctcaagtgc gagaactcgg gcgtggtcaa ggccgtcatc 180 aacaacccgt ttccgttcat tgcggaggtc tcgatcctgg agacgagcaa tggcaaggag 240 aagggcgatc agaaggtcgt gaccagcatc aagccgatgc aacgcgtcgt cgaaggcgac 300 gccaagaagg gcgcgtgatg tacatcgcgc tgtgcatcgt tgcgttgatg ctggtgctga 360 tggtgcagtg ggcgatgcgg tttcagcccg agcagttctg ggcagctgtg gcgaaagagc 420 ctcggtctgc gtgggatcag cacctgaagt ggttgcgcag cgatttcatg catgcgctgc 480 gcatgcgtga tgaggcgtac ctggcgttcg atggtgaggg tcttgagtac gcggatgagt 540 ggttgcgtgc ggacttggac gcgctcggtg gtctagcaaa ggcttggtga tggctcagtg 600 cgtgcagatc gtcggtggtc agtttcagct cgatagcact gcggcatcgt cgtgcacggg 660 ctatctgctg ctgactgctg acgaggtcac gctgctgcat gcgattccgc cgctgtccat 720 tcaggatggg gcaaccatct ctggagcaat cgtcggtgtg tgggcggttg cctggggctt 780 tcgggcagtc gcccgacttt ttttccaacg tgatgaggag gttttatgaa gttcggtatc 840 aagcgtctgg ccgtcgtggc cgctggtgtg ggtggtgccg tggctgctgg cgccgcgaat 900 gctgcgatcg atcttacggc cgtgaccact gcgatcggcg atgctggtac ggccgtgggc 960 acgatcggca gtgccgtgct ggtggtgatc gttggcatcg cggtctacat gtgggtgcgt 1020 cgtcccatca agtgacgctg cggggcgttg tccccgcgcc tggttagacg cgggggctct 1080 ggcccccgtt ttctttggga ggcggtgatg gaaggttact tcgtgatgct cgcgtttttg 1140 ggggcggcat ggattctttt ctcgtgaggg tggtgagcta tgcgctcgtt ctttttctcg 1200 cgtcttgcgc gcatctgggc ggccccccgg gttcttgcgt tactggtttt agcgcttgtc 1260 tgtggcccga gttgggctat tgatccgtcg acgttgggcg ttacggggca agggtggact 1320 gcgacggggc cgaatggtcc gtatttcgat tctgcgggtg cagcttgcgc atctattccg 1380 tctcagtacg tgttgacggg ggtgttggtg cttccgtcgg gtgcggctaa ttgtttggcg 1440 gacattgggg cctattcatc tgtgtttccg gtgggagggc gatgcgcaac gggttatacg 1500 ttgcagtcgg atgggtcgtg tcagaagccg aagccgaatt gtcctgcggc gggtgctgct 1560 ccgagctgga acgttgcgag tggtaagagc cccactgcga cgatcttgaa tcctttgatc 1620 tgcgtaggga actgtgcgta ctcgtatggc agctattgga ccggggcgaa gcctggggcg 1680 ggggggtatg acgcgcaatg gcaggggccg atagcggcga ccggtaatgc gtgcgacggg 1740 agtgagagca acgccagttc gggctcggtg aacgaaaaca gcaacgtcaa ttgcccggac 1800 agaacgtatc agggcacggt gaacgggcaa aacgtctgtg ttccgtatcc gacgcagacg 1860 agtagcggga ccacgacgac gaacactact gatacgcccg ccagcggcgt tccggcgaat 1920 tcgagtagcg cgactacgac gacgacgtgt gatggcacga cctgcacgac aactacgacg 1980 accacgacaa caggcggtgc cggtggcggg tcaggtggtg ctgccagtgg cgtgacgggg 2040 gctagtggtg tggctggcag tgcttgtgtg ggtggcgcga gtgcggttgc tggcacgtgt 2100 acgaccacga cgacacagcc tcagacggat tactgcaagg agaacccgag tgcgtctgtg 2160 tgcgcgagtt cagcgagcgg tggtgcggat tgctcgtcgc cgccgacctg cagcggagat 2220 gcgatcagct gcgcgatcct gagccagcag tggcagacac ggtgcgattt gcagaaggat 2280 gcggattcgt ccatcacgct cggcaagcaa ctgcaggcgg gcaacgatcc tatggctgcg 2340 cagcttccga caccgcagaa ggccgatcag agtcccattg cgatcggaga caagttgtcg 2400 agcgttgaca acatgggcat tgccgagcag tgcctgcagc cgatctcgtt ttcggtctat 2460 agccacacgt acacgttcga cactgggccg atgtgcaagc tcggtcaggt gctgggggcc 2520 ttgaacatca tgagcacgtt gatgctctgc gcttacatgc tgaaggggag tttctgatgc 2580 cattcgttgc gttgctggct tcggccatcg ttggatttct cgcgcaggct gccgtctcgc 2640 tggtcgggcg cgtgctgatc gcgctcggga ttggctttgt cacctacaac gggctcgact 2700 tgttgatgag cggcatcaag tcgctgttcc tgtcgaatgt cagccaggtt gggagcgtgt 2760 tcccgcagat cgttggtgtc atcggcctgc tgaagattcc gatgtccatg aacatgattc 2820 tgaccacgtt ggcgatccgc gccacgctgg ccggcgtgac cggtgggtcg gttcgcaaaa 2880 tgatccagaa gtaggggagg ttgcatgctt acgctcatta ctggccagcc tggcaacggc 2940 aaaagcttgt acgtgatcgc gctggtggag gcgatgcgta aggctgagag ccgcccggtt 3000 ttctactacg gcatcccaga gctgacgctg ccgtggacgc tcctggagga ccccacgacc 3060 tggcatgagt gcccggagaa atcgatcatc gtgatcgatg aagtgcagaa ggtcatgccg 3120 ccgaggccgt cgagctcgaa gccgccggcg catgtggctc cgctggaggt gcatcgccat 3180 cgaggcttcg acctgttctt catgacgcag gatcccagcc tggtcgacaa ccacatcaag 3240 aagttggccg gcgagcatgt ccatctgatt cggcagtttg gcatgcagcg tgcggatgtc 3300 ttcaagatgc agaaggtcca ggatccgacg aacgccaacc tgaagcgtgc gttgcgcacg 3360 acgttcaagt atccgaagga ggtgtttggc tggtacaaga gcgcggacgc ccacacgcac 3420 aagcgcaagg tgccggcgcg tgtgtatttg ctgctggtgc tgccgatttt gctgatcgcg 3480 ttgatctggt tcggagtgaa aacgctgcat ggtgtcggca aggggaagac cgaagcggct 3540 gcgggcgcgc caggcacacc aggtgcaggg ctgcctggcg cttcaactgg tgcacctggt 3600 cggcagaacc aggtgcagac gccagctgag tatctggcta gctatacgcc gcgcgttgcg 3660 gggttgcagt acacggcgcc ggcctatgac gagctcacga agccgtcgcg ggtcccggtg 3720 cctgctgcct gtgtgcagat ccgcgggggc tgtgagtgct ggacccagca gggtacgcac 3780 ttggagacga cgcagcagat ctgcgaccag gtggtgaagc gtggcttctt cgaggccttc 3840 gatgcggatg gtcgggtggc taggaaccag gagcgtgttc aggtcgcgca gcagcccgct 3900 tctgtgcctg ttgcacagcc tcaggaggtg cgtatggttc tgggcgttgc catgccgcgg 3960 gaaacgaccc aggagcctcg caagcgcacg gtggtgggtt cgggcagggc gcgccgtgtt 4020 gacgagctgg atgccttccc agagggctga ttattcgtta cgggtaaaag tgtaacaata 4080 atttaatatt cgttacgagt aatataaaat agagtcatca agtcgggagg cgaatatgcg 4140 tgacgtaacg gataacgtga ctggcgaact cgcgccaggc cgatctggcg aactggtatc 4200 gctgcgaggg cgcacgctga tgcatcccgt ccttagcgcg ctcgtttgtt tcggcttcct 4260 tgcgctttgc gctgcggctg cgttgcgcat tgctggttat tcctggggct tgttctgatg 4320 cgcgtccgtc ccacctcagc ctgtcatctc gatcccacga gcgcggccta tgccgtgcat 4380 ggcacgtccg tccgtgcgcc ggccgacggc tgcgctgacg cgcaccgcgc ggagcgtagc 4440 gacgcgcgag ggcgcggcag cgcttccccc cggcgcggca gcgcttcccc ccgtccggta 4500 atacgggggg aaagtaccga acgcgaggcg ggagccatcg tggactggtt gcgttttacg 4560 ttcctgccgg acggcagcat cggcgacgcc ttggagcatt tgcgtcggta tttccacctg 4620 tggttctcga tccctgtgac gatgaagccg agcgtgcgcg gcttccgggg ctatgagttc 4680 agccacgacc tgctggcgtt cgtcaacggc gagacgatcc gcctgggcat cgtggcgtgt 4740 ggcggtgaga acgtcggcgg gaccatgctg gtagatctgt cggggcaggg ctgtgtcgtg 4800 gtacgcgact ggatggcggt cttcgccacg atgcaggacc tcgatgcgcg tatcacgcgc 4860 tgcgacctgg ccgtggactt ctgccagggt gaggtgacca tcgagcaggt cgagcagatg 4920 tatttcgacg gcgagttcaa cgcgggcggt cgcatcccga agtaccgccg cgtggagagc 4980 ggcgtggcca acgccgaggc ctcgggcggc cggacgttcg agatcggtag gcgcgtgaac 5040 ggcaagctgc tgcgggccta cgagaagggt cgtcagctcg gcaagcagga tagcgactgg 5100 ctgcgcatcg agattgagtt tggcaacaag gatcgcgtga tcccgcatga gattgtgctc 5160 aagcgcgatc actactttgc cggggcgtac aaggcccttg aggcgttcat agcggcggat 5220 ccgcaacggg tcccgactga ccagcgcgac gcgctggagc agcaggacgc catcgtccgc 5280 gagcgcaagc tcgcccatat gaagcagcag ttcggcccga ccgtggacta cgaactgcgc 5340 agcaccaacg aagacttcgc cgcactggtc gttgcgatcc gccgtcaagg ggttcccgcc 5400 cagctgcaca aatctgctct ggcaaagcac gtgtatggcg cgcacgaccc tgtgccgaga 5460 cctgaggagt aagca 5475

Claims (11)

Bacteriophage with specific lytic activity against Ralstonia solanacearum . The bacteriophage of claim 1, wherein the bacteriophage is bacteriophage PE226 (Accession Number: KACC 97007P). The bacteriophage of claim 1, wherein the bacteriophage has a genome consisting of the nucleotide sequence of SEQ ID NO: 1. The bacteriophage of claim 1, wherein the bacteriophage has activity at 25 ° C. to 60 ° C. 7. The bacteriophage of claim 1, wherein the bacteriophage has an activity at pH 4-11. Ralstonia, Ralstonia comprising the bacteriophage of any one of claims 1 to 5 as an active ingredient ( Ralstonia) solanacearum ) Composition for the control of inducible plant diseases. The composition of claim 6, wherein the Ralstonia solanaserum-inducing plant disease is a plant foot blight. 8. The composition of claim 7, wherein the plant is a dicotyledonous plant. A method of controlling Ralstonia solanacearum- induced plant diseases, comprising treating a plant with a composition comprising the bacteriophage of claim 1 as an active ingredient. 10. The method of claim 9, wherein the Ralstonia solanaserum-inducing plant disease is a plant foot blight. The method of claim 10, wherein the plant is a dicotyledonous plant.

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