KR102025403B1 - Novel bacteriophage ΦEaP-8 effective for fire blight bacteria and use thereof - Google Patents

Abstract

The present invention relates to a novel bacteriophage and uses thereof and, more specifically, to an isolated bacteriophage having accession number KFCC11769P and having a lytic activity specific to Erwinia amylovora; an agricultural chemical composition comprising the bacteriophage as an effective component; an antibacterial agent comprising the bacteriophage as an effective component; a disinfectant or detergent comprising the bacteriophage as an effective component; a method for lysing bacteria, comprising the step of treating with the bacteriophage; and a method of controlling a plant disease, comprising the step of treating with the bacteriophage. The novel bacteriophage of the present invention have a lytic activity specific to Erwinia amylovora, and have excellent acid resistance, heat resistance, and UV resistance, and thus can be used in the prevention or treatment of fire blight of fruits caused by Erwinia amylovora, and furthermore, can be widely used in antibacterial agents, disinfectants, detergents, or the like.

Description

Novel bacteriophage ΦEaP-8 effective for fire blight bacteria and use according to novel fruit bacteriophage

The present invention relates to a novel bacteriophage ΦEaP-8 and its use, which is effective against fruit-burn disease bacteria, specifically, bacteriophage isolated with the accession number KFCC11769P having a specific killing ability to Erwinia amylovora ( Erwinia amylobora ), the bacteriophage Agrochemical composition comprising as an active ingredient, an antimicrobial agent comprising the bacteriophage as an active ingredient, a disinfectant or cleaning agent containing the bacteriophage as an active ingredient, a method for lysing bacteria comprising the step of treating the bacteriophage, and the bacteriophage It relates to a method for controlling plant diseases comprising the step of treating.

Erwinia amylovora ( Erwinia amylovora ) is a Gram-negative phytopathogen that causes burn disease in fruit trees such as pears or apple trees. The most common symptom of Erwinia amylobora is that the leaves wither and turn black and dry, and the stems and fruits are initially immersed, pitted, and withered and blackened as if burned. Erwinia amylobora was first discovered in the United States in 1780 and has been reported worldwide in Europe, North America, the Middle East, Central Asia and New Zealand (van der Zwet et al. 2012). The pathogen was first reported in Korea in 2015 through apple and pear burns (Myung et al., 2016; Park et al., 2016). Erwinia amylobora is infected by insects and rainstorms and invades the host through flowers, honey glands, pores, trees, wounds, etc., and is highly contagious and can invade over 70 plants, including pears, apple trees, and apricot trees. It is characterized by the fact that it is causing damage to burn disease worldwide.

Burn disease not only spoils the harvest but can also cause long-term blows. Infection of the flowers causes the fruit to dry and kill, reducing the harvest of the egg and burning the young egg reduces the likelihood of the next year's fruit tree opening. In the roots and trunks of many fruit varieties, burns can destroy large branches or even entire trees. Given the potential impact of burns on fruit harvesting, solutions to the disease are urgently needed.

One method for combating burn disease is to minimize disease outbreak by horticultural practice. For example, there are ways to control infection and spread of burns by reducing soil moisture and balancing fertilizer nutrients. However, this method cannot eliminate the inherent disease outbreak.

Alternatively, it is possible to cure burn disease by removing infected fruit trees or blackened branches in the winter, when the pathogens have a rest period. However, these methods should be careful to sterilize equipment to prevent the spread of disease. This method also cannot completely eradicate the disease because it cannot detect small lesions or internal infections.

In addition, fruit trees infected with burn disease can control burn disease by regularly spraying copper compounds or antibiotics. However, the use of copper compounds is often difficult to use universally because they are often ineffective or cause russeting of fruits. The use of antibiotics, in particular streptomycin, is more effective than copper compounds and is less harmful to fruits. However, Erwinia amylobo is developing resistance to streptomycin in many states in the United States, where California, Oregon, Washington, Missouri, and Michigan are being used, and other methods are needed.

Under these circumstances, the present inventors have made intensive studies to develop a control method for Erwinia amylobora. As a result, bacteriophage isolated from apple orchards in Korea shows lytic activity against Erwinia amylobora, and various environmental factors (temperature , pH, UV) showed that the bacteriophage can be used as a phage therapy for burn disease in fruit crops, to complete the present invention.

One object of the present invention is to provide an isolated bacteriophage having accession number KFCC11769P, which has specific killing ability to Erwinia amylovora ( Erwinia amylobora ).

Another object of the present invention to provide a pesticide composition comprising the bacteriophage as an active ingredient.

Yet another object of the present invention is to provide an antimicrobial agent comprising the bacteriophage as an active ingredient.

Still another object of the present invention is to provide a disinfectant or cleaning agent comprising bacteriophage as an active ingredient.

Yet another object of the present invention is to provide a method for lysing Erwinia amylovora ( Erwinia amylovora ) comprising the step of treating the bacteriophage.

Yet another object of the present invention is to provide a method for controlling fruit burn disease, comprising the step of treating the bacteriophage.

One aspect of the present invention for achieving the above object provides an isolated bacteriophage having accession number KFCC11769P, which has specific killing ability to Erwinia amylovora ( Erwinia amylobora ).

In the present invention, the term “bacteriophage” is a virus that infects a particular target bacterium. The head of a bacteriophage wrapped in a protein capsid consists of the nucleic acid genome, and some bacteriophages have tails composed of lipids or proteins. Most bacteriophages contain double-stranded DNA as genetic material.

More than 95% of the known bacteriophage belongs to kawoodo Bilal less (Caudovirales) neck (order) (Maniloff J and Ackermann HW 1998. Arch Virology 143:... 2051-2063.). The three major families of bacteriophages are distinguished by their morphological features, particularly by their tails. Phages belonging to the Myoviridae family have a bilayer contractile tail, phages belonging to the Siphoviridae family have long elastic tails, and phages belonging to the Podoviridae family have short, blunt tails. (Deveau H, 2006, Appl. Environ. Microbiol. 72: 4338-4346.).

Bacteriophages can be found in water, in soil, and in any environment inside or outside plants. Bacteriophages attach to target bacteria and inject their DNA into the host, and self-replicate in the host causing the death of the bacteria. Bacteriophages are relatively safe for animal or plant cells (Kutter E and Sulakvelidze A. 2004. CRC Press.) And do not affect other beneficial microorganisms. In addition, bacteriophages are relatively easily separated from the host bacterial environment.

Recently, bacteriophages are plant pathogenic bacteria, for example Dickeya solani (Czajkowski R, 2014, Plant Pathol. 63: 758-772.), Pectobacterium carotovorum (Lim JA, J. Microbiol. Biotechnol. 19 23: 1147-1153.) Or as a substance of phage therapy for controlling plant diseases caused by Ralstonia solanacearum (Young BJ, J. Microbiol. Biotechnol. 22:16 1613-1620.). However, due to the genetic diversity of bacteriophages, it is necessary to identify specific bacteriophages with specific bacterial specific killing ability for controlling plant diseases.

On the other hand, the bacteriophage of the present invention is a novel phage having a genomic sequence different from the known phage (FIG. 9), and was first identified by the present inventors as having specific killing ability to Erwinia amylobora.

We collected soil samples from apple orchards in Korea and isolated bacteriophages that have specific killing capacity for Erwinia amylobora. In addition, as a result of observation through a transmission electron microscope, it was confirmed that the bacteriophage belongs to the Podoviridae family (Fig. 4).

In addition, the bacteriophage investigated the stability in the temperature and pH range, not only stably survive in the temperature range up to 50 ℃ and pH range of pH 5 to 10, but also exhibits the characteristics that can maintain stability even when exposed to ultraviolet light It was confirmed (FIGS. 6-8).

Such bacteriophages having specific killing ability to Erwinia amylobora and having stability exhibit an effective antibacterial activity against Erwinia amylobora, and thus may have a useful effect in the control of fruit tree burn disease.

Accordingly, the present inventors have a specific killing ability in Erwinia amylobora and the bacteriophage having the above characteristics April 19, 2018 Korean Culture Center of Microorganisms, 361-221 Hongje 1-dong, Seodaemun-gu, Seoul Deposited as KFCC11769P.

In the present specification, the bacteriophage deposited with the accession number KFCC11769P may be referred to as ΦEaP-8 or Φ8.

Another embodiment of the present invention provides a pesticide composition comprising the bacteriophage as an active ingredient.

At this time, the definition of the bacteriophage is as described above.

As used herein, the term "pesticide" refers to a medicament used for the protection and growth of crops, and chemical pesticides, biopesticides, crops used to control pests, pests, weeds, and the like, which occur during the cultivation or storage of crops. Growth regulators, used to enhance or inhibit the physiological function of the drug, and the like to improve the drug generically.

Since the pesticide composition of the present invention contains bacteriophage having the killing ability specific to Erwinia amylobora as an active ingredient, the pesticide composition may be used for the prevention or treatment of fire blight.

Specifically, the pesticide composition may be an intraocular injection composition, a biologic composition, a fertilizer composition, a nutrition composition, a plant fortification composition, a plant protection composition, a soil conditioner composition or a yield enhancer composition, It is not limited to this.

Formulation of the agrochemical composition may be a wettable powder, a water dispersible granule, a dusting powder, a liquid concentrate, an emulsion, a tablet, a liquid, or It may be formulated as an oil suspension, but is not limited thereto.

The pesticide composition may comprise one or more dispersants, with an effective amount of water, which may be included in the range of about 2 to about 60% (w / w). Various dispersants sold by Rhone Poulenc, Witco, Westvaco, International Specialty products, Crodachemicals, Borregaard, BASF, Rhodia, and the like are commercially available for use in agrochemical compositions.

Specific examples of dispersants that may be used in the agrochemical composition include polyvinylpyrrolidone, polyvinyl alcohol, lignosulfonate, phenyl naphthalene sulfonate, ethoxylated alkyl phenols, ethoxylated fatty acids, alkoxylated linear alcohols, rings Aromatic (polyaromatic) sulfonates, sodium alkyl aryl sulfonates, glyceryl esters, maleic anhydride copolymers, phosphate esters, condensation products of aryl sulfonic acids and formaldehyde, condensation products of alkylaryl sulfonic acids and formaldehyde, ethylene oxide Addition reaction products of fatty acid esters with, salts of addition reaction products of ethylene oxide and fatty acid esters, sulfonates of condensed naphthalene, addition reaction products of ethylene oxide and fatty acid esters, addition of ethylene oxide and fatty acid esters Salts of reaction products, lignin derivatives, naphthalene po Sodium aldehyde condensate, sodium salt of isodecylsulfosuccinic acid half ester, polycarboxylate, sodium alkylbenzenesulfonate, sodium salt of sulfonated naphthalene, ammonium salt of sulfonated naphthalene, salt of polyacrylic acid, phenol It may be, but is not limited to, a salt of sulfonic acid or a salt of naphthalene sulfonic acid.

In addition, the pesticide composition may further comprise a humectant, a binder, a filler (filler) or an organic additive.

The wetting agent may be included in the pesticide composition in the range of about 0.1 to 5%, and in particular, alkyl naphthalene sulfonate, sodium alkyl naphthalene sulfonate, sodium salt of sulfonated alkylcarboxylate, polyoxyalkylated ethyl phenol, polyoxy Polyoxyethoylated fatty alcohols, polyoxyethoxylated fatty amines, lignin derivatives, alkanesulfonates, alkylbenzene sulfonates, salts of polycarboxylic acids, salts of esters of sulfosuccinic acids, alkylnaphthalenesulfonates, alkylbenzenesulfonates , Alkylpolyglycol ether sulfonate, alkyl ether phosphate, alkyl ether sulfate or alkyl sulfosuccinic acid monoester, but is not limited thereto.

The binder may be included in the pesticide composition in the range of about 0.1 to 7%, specifically, polyvinyl alcohol, lignin derivatives, polyvinyl pyrrolidone, polyalkylpyrrolidone, carboxymethylcellulose, xanthan gum, poly Ethoxylated fatty acids, polyethoxylated fatty alcohols, ethylene oxide copolymers, propylene oxide copolymers, polyethylene glycols or polyethylene oxides, but are not limited thereto.

The filler may be included in the pesticide composition in the range of about 0.1 to 10%, specifically, bentonite, sub-bentonite, attapulgite, kaolinite, montmorillonite, bauxite, Hydrated alumina, calcined alumina, diatomaceous earth, chalk, fuller's earth, dolomite, kieselguhr, loess, prophyllite, Talc, vermiculite, limestone, natural and synthetic silicates, silica or china clay, but are not limited thereto.

The organic additive may be included in the pesticide composition in the range of about 0.01 to 50%, for example, macronutrient, micronutrient compost fertilizer, natural element, natural organism organisms, trichoderma, humic acid extracts, Bacillus thuringiensis, viruses, natural fungi, plant extracts, pesticides, biological control products, natural oils, natural Extract, mineral or urea group, but is not limited thereto. The micronutrient may be zinc, iron, manganese, copper, boron, cobalt, vanadium, selenium, silicon or nickel, but is not limited thereto. The macronutrient may be nitrogen, phosphorus, potassium, calcium or magnesium, but is not limited thereto.

Another embodiment of the present invention provides an antimicrobial agent comprising the bacteriophage as an active ingredient.

As used herein, the term "antimicrobial agent" means a substance that kills microorganisms or inhibits development, and generically refers to preservatives, fungicides and antibiotics.

The bacteriophage of the present invention not only exhibits high antimicrobial activity specifically against Erwinia amylobora, but also kills Erwinia pyripolyae, and has excellent heat resistance, acid resistance and ultraviolet resistance. Therefore, the antimicrobial agent containing the bacteriophage as an active ingredient is safe because it does not induce drug resistance or resistance as compared to the existing antimicrobial material, and can be used as a new antimicrobial agent with a long life cycle.

Another aspect of the invention provides a disinfectant or cleaning agent comprising the bacteriophage as an active ingredient.

The term "disinfectant" in the present invention means a medicament for the purpose of sterilizing pathogens, and the term "cleaning agent" in the present invention means a medicament used for the purpose of cleaning.

The bacteriophage of the present invention not only exhibits high antimicrobial activity specifically against Erwinia amylobora, but also kills Erwinia pyripolyae, and has excellent heat resistance, acid resistance and ultraviolet resistance. Therefore, the disinfectant or cleaning agent containing the bacteriophage as an active ingredient has excellent sterilizing activity as compared to existing drugs, and can be used as a new disinfectant or cleaning agent having a long life cycle.

Yet another aspect of the present invention provides a method for lysing Erwinia amylobora comprising treating the bacteriophage.

As used herein, the term "lysate" refers to a phenomenon in which a cell wall or cytoplasmic membrane of a bacterium is destroyed to dissolve the bacteria, and a phenomenon in which the bacteria are killed.

Specifically, the method for lysing the bacteria may include, but is not limited to, treating the bacteria with an effective amount of a pesticide composition, an antimicrobial agent, a disinfectant or a cleaning agent comprising the bacteriophage of the present invention as an active ingredient.

Yet another aspect of the present invention provides a method for controlling a fruit tree disease comprising treating the bacteriophage.

As used herein, the term "fruit tree disease" is a plant disease caused by Erwinia amylobora, which occurs in flowers, leaves, branches, fruits and stems, and is particularly prevalent in new branches. It occurs at the edges of the first leaves and progresses along the leaf veins, leaving the leaves withering and turning black. The flower is initially immersed in water, and withered, turning dark brown, falling or hanging. Onset of a new branch starts at the end and progresses downwards, suddenly withering away. Stems and fruits also initially immersed in lesions, pitted, and withered. In addition, it is contagious because it is transmitted by insects or rainstorm and invades the host through flowers, honey glands, pores, trees, wounds, and the like.

As used herein, the term "control" means preventing and controlling various pests that damage crops.

Specifically, the method for controlling the fruit tree disease comprises the step of treating the effective amount of the pesticide composition, antimicrobial, disinfectant or cleaning agent containing the bacteriophage of the present invention as an active ingredient to the basal or leaf part of the plant It may be, and more specifically may include the step of treating the diseased part of the plant, but is not limited thereto.

The novel bacteriophage of the present invention has specific killing ability to Erwinia amylovora ( Erwinia amylobora ), and is excellent in acid resistance, heat resistance, and UV resistance, and thus, prevention of fruit tree disease caused by Erwinia amylovora or Not only can be used for treatment, it can also be widely used in antibacterial, disinfectant, cleaning agent and the like.

1 is a view schematically showing the process of separation, growth and purification of bacteriophage.
2 is a diagram showing restriction enzyme cleavage patterns of 21 bacteriophages isolated.
3 shows restriction enzyme cleavage patterns of selected three bacteriophages.
Figure 4 is a view showing the shape of the bacteriophage confirmed by transmission electron microscope.
5 is a diagram showing the lytic activity of bacteriophage against Erwinia genus strains.
6 is a graph showing the results of stability analysis of bacteriophages at various temperatures.
7 is a graph showing the results of stability analysis of bacteriophage against 365 nm UV exposure.
8 is a graph showing the results of stability analysis of bacteriophage against various pH values.
9 is a diagram illustrating a result of comparing genome information of three bacteriophages with genome information of existing bacteriophages.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Test Method 1. Bacterial Strain and Soil Sampling

Bacteriophages are present in nature, such as plants, in which surrounding soil, water and host bacteria may be present. So we collected apple and pear orchard soil for bacteriophage separation. Soil samples were taken from apple orchards at Jecheon, Chungju, and Kyunghee University. A total of 18 soil samples were divided into three groups belonging to the same area and screened for the isolation of bacteriophages against 12 Erwinia amylobora and three Erwinia pyrifoliae strains.

Experimental Method 2. Isolation and Purification of Bacteriophage

The soil samples were mixed and shaken for 30 minutes to allow the bacteriophage to be well extracted with distilled water. The mixed samples were centrifuged (10,000 rpm, 10 min, 4 ° C.) and filtered (0.22 μm pore size filter; Sartorius, Germany) to obtain supernatant. Bacteriophage was concentrated by incubating overnight in 5 ml of supernatant, 10 ml of LB and 500 ul of bacterial suspension (10 ⁹ CFU / ml) in a 26 ° C. shaker incubator. The cultured cells were treated with chloroform (1% of the final volume) for 30 minutes to remove host bacteria and residues, and then centrifuged (3,000 g, 15 min, 4 ° C.) and filtered (0.22um pore size filter, Futecs, South Korea). ). Bacteriophage concentrates were grown in four selected Erwinia amylobora strains and two Erwinia pyripolyea strains and confirmed the presence of bacteriophage using a dotting assay. To separate the bacteriophages, plaque picking (Vidaver et al., 1973; Vidaver and Schuster, 1969), with different sizes and shapes of bacteriophages, was repeated at least five times.

Bacteriophage was purified via CsCl gradient ultracentrifugation. CsCl gradient ultracentrifugation consisted of four densities of CsCl layers (1.3 g / ml, 1.45 g / ml, 1.5 g / ml and 1.75 g / ml). The bacteriophages and samples were ultracentrifuged (25,000 rpm, 2h, 4 ° C.) and the bacteriophages were located at CsCl densities of 1.45 g / ml and 1.5 g / ml. Bacteriophage was harvested and purified after dialysis (1 hour, 2 times) using sodium chloride-magnesium sulfate (SM) buffer (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgSO ₄ ). The schematic isolation, propagation and purification of bacteriophages is shown in FIG. 1.

Experiment Method 3. Spotting and Overlay Assays

Spotting assays were performed with minor modifications to known methods (Kropinski et al., 2009; Yu et al., 2016). Host bacteria were incubated overnight, then 200 μl (10 ⁹ CFU / mL) of the sample was mixed with 5 mL of soft LB agar (0.4% agar). The mixture was poured into a prepared LB solid medium plate and dried at room temperature. Thereafter, 10 μl of bacteriophage stock dilution was dropped on the agar layer and the plate was dried at room temperature for 30 minutes. The culture was incubated overnight at 26 ° C. and plaques were examined the next day.

Overlay assays were performed with minor modifications to known methods (Yu et al., 2016). 0.4 μl of soft LB agar (0.4% agar) and 200 μl of overnight bacterial culture (10 ⁹ CFU / ml) and 100 μl of diluted bacteriophage (10 ⁴ PFU / ml) were mixed and the solid LB medium The mixture was poured and left to dry at room temperature for 30 minutes. The plates were incubated at 26 ° C. and plaques were examined the next day.

Experimental Method 4. Growth of Bacteriophage

Proliferation of bacteriophages was carried out with minor modifications to known methods (Kim and Ryu, 2011). A single colony of Erwinia amylobora mixed with LB liquid medium was incubated in a shaker overnight at 26 ° C. A lysate of bacteriophage was added to the cultured Erwinia amylobora bacterial suspension (OD ₆₀₀ = 0.5-1.0) and incubated with a shaker overnight at 26 ° C. The cultured samples were centrifuged (10,000 rpm, 10 min, 4 ° C.) and filtered (0.22um pore size, Sartorius, Germany). 30 ml of the filtered sample, 5 g of PEG 6000, and 10 ml of 5 M NaCl were shaken for 30 minutes, mixed and incubated overnight in a 4 ° C. refrigerator. The cultured samples were centrifuged (9,000 g, 20 min, 4 ° C.), then the supernatant was discarded and the pellet was dissolved in 3 ml of SM buffer.

Experimental Method 5. Morphological Analysis of Bacteriophage

Phage particles were stained by adding a carbon coated copper grid with purified bacteriophage stock and 2% aqueous uranyl acetate (pH 4.0) for 30 seconds (Ackermann and Heldal, 2010; Brum and Steward, 2010). Five bacteriophages (Φ7, Φ8, Φ19, Φ20, and Φ21) were identified using a 120 kV transmission electron microscope (TEM) at the Korea Basic Science Institute (KBSI). Bacteriophages were classified according to the criteria of the International Committee on Taxonomy of Viruses.

Experimental method 6. Bacteriophage DNA extraction and restriction enzyme digestion

21 isolated bacteriophages were extracted from genomic DNA using a phage DNA separation kit (Norgen Biotek, Canada). Restriction digestion was performed using 3 μl of bacteriophage DNA with enzyme (EcoRI and BamHI, or both) at 37 ° C. for 2 hours. Cleaved bacteriophage DNA samples were examined by gel electrophoresis containing 0.7% agarose containing EcoDye.

Test Method 7. Stability Analysis of Bacteriophage at Various Temperatures, pH and UV Conditions

Three bacteriophages were examined for stability under various conditions such as temperature, pH and UV conditions. In the case of temperature, it processed for 1 hour at 30 degreeC, 40 degreeC, 50 degreeC, and 60 degreeC. For pH, the pH conditions were treated for 1 hour in the pH range of 3-12 adjusted to HCl and NaOH. For UV, UV conditions of 365 nm were treated for 1 hour and measured every 15 minutes. The concentration of bacteriophage used in the experiment was 10 ⁵ PFU / ml. The remaining bacteriophages were measured for the dotting assay.

In the above, the contents confirmed through the experimental method are summarized in the following examples.

Example 1 Isolation of Bacteriophage in Apple Orchard Soils

A list of bacteriophages, including information about host bacterial strains and isolated sites, is in Table 1. Soil samples from all apple orchards except Kyunghee University's apple orchards were infected with Erwinia amylobora. 21 isolated bacteriophages were observed through plaque size and shape. When classified into separate sites, Jecheon separated four bacteriophages, Chungju separated only one bacteriophage, and 16 bacteriophages from the apple orchard soil of Kyung Hee University. When classified into host bacterial strains, seven bacteriophages were isolated from Erwinia amylobora strain 73, ten bacteriophages were isolated from Erwinia amylobora strain Ea-k1 (79), and Erwinia pyrifolia strain 81 Three bacteriophages were isolated from, and one bacteriophage was isolated from Erwinia pyripolyea strain Epk1 / 15 (81).

Phage No. Host Strain Soil sampling sites One E. amylovora 73 Kyung Hee University 2 E. amylovora 73 Kyung Hee University 3 E. amylovora 73 Kyung Hee University 4 E. amylovora 73 Kyung Hee University 5 E. amylovora 73 Kyung Hee University 6 E. amylovora 73 Kyung Hee University 7 E. amylovora 73 Jecheon 8 E. amylovora Ea-K1 (79) Kyung Hee University 9 E. amylovora Ea-K1 (79) Kyung Hee University 10 E. amylovora Ea-K1 (79) Kyung Hee University 11 E. amylovora Ea-K1 (79) Kyung Hee University 12 E. amylovora Ea-K1 (79) Kyung Hee University 13 E. amylovora Ea-K1 (79) Kyung Hee University 14 E. amylovora Ea-K1 (79) Chungju 15 E. amylovora Ea-K1 (79) Jecheon 16 E. amylovora Ea-K1 (79) Jecheon 17 E. amylovora Ea-K1 (79) Jecheon 18 E. pyrifoliae 81 Kyung Hee University 19 E. pyrifoliae 81 Kyung Hee University 20 E. pyrifoliae 81 Kyung Hee University 21 E. pyrifoliae Epk1 / 15 (81) Kyung Hee University

Example 2 Determination of Host Range

To determine the host range for various bacteria, five representative bacteriophages (Φ7, Φ8, Φ19, Φ20, Φ21) were subjected to a host range test using an overlay assay. The host range of three selected bacteriophages (Φ7, Φ8, Φ21) was 20 Erwinia amylobora strains, 8 Erwinia pyrifolia strains, Pectobacterium carotovorum subsp. Carotovorum , and Dickeja Dickeya Zeae and three Pantoea strains (formerly Erwinia strains) were determined. Regardless of where it was isolated, all three bacteriophages were found to be effective only in Erwinia amylobora and Erwinia pyrifolia strains (Table 2).

Example 3. Genomic DNA Pattern Analysis of Purified Bacteriophage

In order to confirm whether the separated bacteriophages are identical, the genomic DNA of the isolated bacteriophages was extracted through experiment 6 and restriction enzyme cleavage was performed to compare the genomic DNA patterns of the bacteriophages with the restriction enzyme cleavage results. The 21 bacteriophage DNA patterns were of three types, either cutting only EcoRI, cutting only BamHI, or cutting EcoRI and BamHI (FIG. 2). Representative bacteriophages of each group were selected as Φ 7, Φ 8, and Φ 21 according to the DNA pattern (FIG. 3). Genomic DNA of all bacteriophages was digested with EcoRI, but only one bacteriophage (Φ8) was digested with BamHI. The estimated sizes of the genomic DNAs of bacteriophage? 7,? 8, and? 21 were about 70 kb, 75 kb, and 80 kb, respectively.

TABLE 2-1

Table 2-2

Example 4 Morphology of Bacteriophage

To characterize and classify bacteriophages, the morphology of the purified bacteriophages was determined. Three selected bacteriophage morphologies were identified using transmission electron microscopy. As a result, it was confirmed that Φ7, Φ8 and Φ21 belong to the Caudovirales throat, Φ7 and Φ21 belong to the Myoviridae family and Φ8 belonged to the Podoviridae family (Fig. 4). The head length of Φ7 was 88.8nm, the head length of Φ8 was 75nm, and the head length of Φ21 was 63nm.

The bacteriophage Φ8 was deposited on April 19, 2018 with the deposit number KFCC11769P at the Korea Microorganism Conservation Center.

Example 5 Analysis of Lysis Activity of Bacteriophage

The lytic activity of bacteriophage was analyzed as the most important factor that can be applied as phage therapy for controlling plant diseases.

Bacteriophage lysates were inoculated into Erwinia amylobora or Erwinia pyripolyea obtained by the method of Experiment 4, and cultured at 26 ° C. to analyze lytic activity.

As a result, as shown in Figure 5, bacteriophage Φ7, Φ8, Φ19, Φ20, Φ21 was confirmed that all showed excellent lytic activity against Erwinia amylobora or Erwinia pyripolyae.

Example 6 Stability of Bacteriophage to Temperature, pH and UV

In order to use bacteriophage as an alternative, stability of various environmental conditions such as temperature, soil pH and ultraviolet light is important. Accordingly, after treating each environmental condition, a dotting assay was performed to confirm viable bacteriophages (FIGS. 6, 7 and 8). In the case of temperature conditions, Φ 7 and Φ 8 were stable up to 50 ° C. for 1 hour, but the viability began to decrease above 50 ° C. and was completely inactivated at 60 ° C. Φ21 was stable up to 40 ° C. and viability began to decrease above 40 ° C. and completely deactivated at 60 ° C. For pH conditions, Φ7 and Φ21 were stable at pH 4-11 and Φ8 was stable at pH 5-10. In the case of UV, UV at 365 nm was stable in all three bacteriophages.

Through the above results, it can be seen that the bacteriophage of the present invention is stable to various temperatures, various pH ranges and ultraviolet rays.

Example 7 Genomic Information Analysis

After genome information was obtained from bacteriophage DNA extracted through Experiment 6, it was compared with genome information of known Erwinia specific bacteriophage. As a result, Φ8 showed a lower sequence identity than Φ7 or Φ21, and Φ8 and Φ21 had low homology with previously reported bacteriophages, confirming that they were novel bacteriophages (FIG. 9).

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

[Accession number]

Name of Depositary: Korea Microorganism Conservation Center (Domestic)

Accession number: KFCC11769P

Deposit Date: 20180419

Claims (9)

An isolated bacteriophage with Accession No. KFCC11769P, with specific simultaneous killing ability to Erwinia amylovora ( Erwinia amylobora ) and Erwinia pyrifoliae ( Erwinia pyrifoliae ).
A pesticide composition comprising the bacteriophage of claim 1 as an active ingredient.
The pesticide composition of claim 2, wherein the bacteriophage is used for the prevention or treatment of fire blight.
3. The pesticide composition of claim 2, wherein the pesticide composition is an intraocular injection composition, a biologic composition, a fertilizer composition, a nutrition composition, a plant fortification composition, a plant protection composition, a soil conditioner composition, and a yield enhancer composition. At least one selected from the group consisting of, pesticide composition.
An antimicrobial agent comprising the bacteriophage of claim 1 as an active ingredient.
Disinfectant comprising a bacteriophage of claim 1 as an active ingredient.
A method of simultaneously lysing Erwinia amylovora ( Erwinia amylobora ) and Erwinia pyrifoliae ( Erwinia pyrifolia ) comprising the step of treating the bacteriophage of claim 1 to bacteria.
A method of controlling fruit tree disease comprising treating the bacteriophage of claim 1 to a plant.
A detergent comprising the bacteriophage of claim 1 as an active ingredient.

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