Bacteriophage Fifi011 for controlling fire blight and black blight

Abstract

The present invention relates to a novel bacteriophage Fifi011 having lytic activity specific to fruit tree fire blight pathogens, a composition and pesticide for controlling fruit tree fire blight containing the same, and a method for controlling the same. According to the novel phage of the present invention, it has lytic activity (killing ability) against both Erwinia amylovora and Erwinia pyrifolia, so it can effectively deal with fruit tree fire blight, prevent the emergence of antibiotic-resistant strains, and can be used for safe and excellent control of fruit tree fire blight without affecting the environment.

Description

Technology Field

[0001] The present invention relates to a novel bacteriophage Fifi011 for controlling fire blight and black rot of eggplant, and a control method using the same. Background Technology

[0002] Fire blight is one of the representative bacterial plant quarantine diseases, Erwinia amylovora ( Erwinia amylovora It is known that ) is the causative agent. Fire blight causes the flowers, branches, and fruits of trees such as pear or apple to turn black, resulting in symptoms where they dry up and die black, appearing as if they have been burned. Because *Erwinia amylovo* is transmitted by insects or wind and rain and invades the host through flowers, nectaries, stomata, lenticels, and wounds, it is highly contagious and can affect about 70 species of plants, including pear, apple, and apricot trees, causing damage from fire blight worldwide.

[0003] In addition, black blight is also one of the representative bacterial plant quarantine diseases, and Erwinia pyrifolia ( Erwinia pyrifoliae ) is known as the causative agent. *Erwinia pyrifolia* is *Pyrus pyrifolia* of the Asian pear tree ( Pyrus pyrifolia It causes severe necrosis in ) trees. In 1995, black or brown stripes on the central veins and necrosis of the leaf petioles were observed in the leaves of the Asian pear variety *Shingo* at an orchard near Chuncheon. These symptoms spread to the entire branches and affected flowers and small fruits, resulting in a significant decrease in fruit production.

[0004] Conventionally, methods to address fire blight and black blight in fruit trees have been known, such as controlling the infection and spread of the causative agent by reducing soil moisture and maintaining a balance of fertilizer nutrients, or removing infected fruit trees or blackened branches during the winter when the causative agent is dormant, but these methods could not suppress the occurrence of the disease in essence.

[0005] Accordingly, the inventors studied phage therapy, a control method using bacteriophages, as a control method for Erwinia amylovora and / or Erwinia pyrifolia. As a result, they isolated and characterized a novel bacteriophage Fifi011, which has specific lytic activity against both of the said pathogens, and confirmed its control effect against both fire blight and black blight. Prior art literature

[0006] Korean Registered Patent No. 10-1601912 The problem to be solved

[0007] The present invention aims to provide a novel bacteriophage Fifi011 having lytic activity specific to Erwinia amylovora and / or Erwinia pyrifolia, a composition for controlling fruit trees containing the same, a pesticide, and a method for controlling the same. means of solving the problem

[0008] One aspect provides a novel bacteriophage Fifi011 (accession number: KACC 97043P).

[0009] The above bacteriophage Fifi011 is Erwinia amylovara ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliae It has lytic activity against one or more microorganisms selected from the group consisting of ).

[0010] The above term "Erwinia Amylovora ( Erwinia amylovora )" is the causative pathogen of fire blight, and Enterobacterial, a Gram-negative bacterium ( Enterobacterales It belongs to the order. Meanwhile, the above-mentioned fire blight is one of the representative bacterial plant quarantine diseases and is an infectious disease that affects other plants such as apples, pears, and the rosemary family.

[0011] The above term "Erwinia piripoliae ( Erwinia pyrifoliae"..." is the causative pathogen of black blight and is a Gram-negative bacterium. Meanwhile, the black blight mentioned above is an infectious disease that affects other plants such as apples and pears.

[0012] The term "lysation" above refers to the phenomenon in which microorganisms are dissolved due to the destruction of their cell walls or cytoplasmic membranes, and signifies the phenomenon of microorganisms dying.

[0013] The term "bacteriophage" refers to a virus that infects specific target microorganisms. The head of a bacteriophage, which is packaged in a protein capsid, consists of a nucleic acid genome, while some bacteriophages have a tail composed of lipids or proteins. Most bacteriophages contain double-stranded DNA as their genetic material.

[0014] In one aspect, the bacteriophage Fifi011 is Erwinia amylovara ( Erwinia amylovora ) and has lytic activity against Erwinia pyrifolia, and the above Erwinia amylovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliae It may have a control effect by killing )

[0015] In one embodiment, to confirm the lytic activity of bacteriophage Fifi011, Erwinia amylovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliae Plaque was identified using ).

[0016] As a result, the above bacteriophage Fifi011 is Erwinia amylovora, the causative agent of fruit tree fire blight ( Erwinia amylovora It formed clear plaques and showed strong lytic activity, and Erwinia pyrifolia, the causative agent of black rot of eggplant ( Erwinia pyrifoliae It was confirmed that it forms a transparent plaque and exhibits strong lytic activity (see Example 1).

[0017] In one aspect, the bacteriophage Fifi011 is a grapevine ( Podoviridae It could be family.

[0018] The above term "grape berry family ( Podoviridae "family" refers to Caudovirales ( Caudovirales It is a family of bacteriophages in the order ) and has a polygonal head and a short tail of less than 17 nm. Podoviridae It includes 8 genera assigned to 3 subfamilies and 45 genera not assigned to subfamilies.

[0019] In one embodiment, to analyze the morphology of the bacteriophage Fifi011, the morphology of the phage samples isolated using CsCl density difference was confirmed using a transmission electron microscope. Additionally, phylogenetic analysis was performed through DNA sequencing. As a result, it was confirmed that the bacteriophage belongs to the Podoviridae family (see Examples 2 and 5).

[0020] In another embodiment, the host range of the bacteriophage Fifi011 was determined. As a result, the bacteriophage Fifi011 was found to be Erwinia amylovora, the causative agent of fruit tree fire blight ( Erwinia amylovora ) and Erwinia pyrifolia, the causative agent of black eggplant blight ( Erwinia pyrifoliae It was confirmed that it forms a transparent plaque and exhibits strong lytic activity (see Example 3).

[0021] In one aspect, the bacteriophage Fifi011 may have lytic activity at 10°C to 70°C, 10°C to 65°C, 10°C to 60°C, 15°C to 70°C, 15°C to 65°C, 15°C to 60°C, 20°C to 70°C, 20°C to 65°C, or 20°C to 60°C.

[0022] In one embodiment, changes in activity of the bacteriophage Fifi011 at various temperatures were investigated. As a result, it was confirmed that the bacteriophage Fifi011 exhibited excellent lytic activity even after treatment at 40°C for 1 hour (see Example 4).

[0023] In one aspect, the bacteriophage Fifi011 may have lytic activity at pH 1 to 12, pH 1 to 11, pH 1 to 10, pH 2 to 12, pH 2 to 11, pH 2 to 10, pH 3 to 12, pH 3 to 11, or pH 3 to 10.

[0024] In one embodiment, changes in the activity of the bacteriophage Fifi011 at various pH values ​​were investigated. As a result, it was confirmed that the bacteriophage Fifi011 exhibited excellent lytic activity even when exposed to various pH values ​​from 5 to 10 for 1 hour (see Example 4).

[0025] In one aspect, the bacteriophage Fifi011 may have lytic activity in a concentration range of 0.001 to 0.1 MOI (Multiplicity of infection).

[0026] In one embodiment, even when the bacteriophage Fifi011 is treated at a low concentration of 0.001 to 0.1 MOI (Multiplicity of infection), Erwinia amylovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliea It was confirmed that it has excellent lytic activity against ) (see Example 6).

[0027] Another aspect is Erwinia Amilovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliae A control composition comprising bacteriophage Fifi011 (accession number: KACC 97043P), having lytic activity against one or more microorganisms selected from the group consisting of ).

[0028] The above "Erwinia amylovora," "Erwinia pyrifolia," "lysative," "bacteriophage," etc. may be within the aforementioned range.

[0029] The term "control" above refers to the prevention and eradication of various pests, diseases, or microorganisms that cause damage to crops.

[0030] In one aspect, the above-mentioned pest control composition is Erwinia amylovora ( Erwinia amylovora ) and has lytic activity against Erwinia pyrifolia, and the above Erwinia amylovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliae It may have a control effect by killing )

[0031] In one aspect, when the above-mentioned control composition is treated at a concentration of 0.001 to 0.1 MOI (Multiplicity of infection), Erwinia amylovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliea It was found that the lytic activity against ) was excellent (see Example 5).

[0032] In one aspect, the composition may further include an excipient.

[0033] Since the above excipients are not limited to specific ones used in microbial pesticides, a description regarding the excipients will be omitted. For example, a pest control composition according to one aspect may use surfactants or fillers usable as biochemical pesticides as excipients for the purpose of stable formulation suitable for actual use in the field.

[0034] For example, the above surfactant is an alkyl (C8~C 12 )arylsulfonate, dialkyl (C3~C6)arylsulfonate, dialkyl (C8~C 12 )sulfosuccinate, ligninsulfonate, naphthalenesulfonate condensate, naphthalenesulfonateformalin condensate, alkyl(C8~C12 )Naphthalenesulfonate formalin condensate, polyoxyethylenealkyl (C8~C 12 ) Sodium or calcium salts of sulfonate compounds such as phenylsulfonate, alkyl (C8~C 12 )sulfate, alkyl (C8~C 12 )aryl sulfate, polyoxyethylenealkyl (C8~C 12 )sulfate, polyoxyethylenealkyl (C8~C 12 Sodium or calcium salts of sulfate compounds such as phenyl sulfate, sodium or calcium salts of succinate compounds such as polyoxyalkylene succinate, anionic surfactants such as sodium benzoate and alkyl carboxylates, polyoxyethylenealkyl (C8~C 12 )ether, polyoxyethylenealkyl (C8~C 12 )phenyl ether, polyoxyethylenealkyl (C8~C 12 It may be one or more selected from the group consisting of phenyl polymers, nonionic surfactants such as ethylene oxide propylene oxide copolymers, polycarboxylates, Triton 100, and Tween 80.

[0035] In addition, the above-mentioned fillers may include bentonite, talc, clay, kaolin, calcium carbonate, silica sand, pumice, diatomaceous earth, acidic white clay, zeolite, perlite, white carbon, ammonium sulfate, urea, glucose, dextrin, soybean flour, rice, wheat, red clay, glucose and starch, water, etc., used alone or in a mixture of two or more types.

[0036] In one aspect, the composition may further include an antibiotic. There is no limitation on the type of antibiotic, but it may be streptomycin.

[0037] In one aspect, the composition may be one or more formulations selected from the group consisting of wettable powders, granular wettable powders, liquid wettable powders, liquids, aqueous solutions, water-soluble granules, and encapsulating agents. These may be prepared by known methods, for example, by mixing an active compound with an extender (liquid solvent or solid carrier) using optionally a surfactant (emulsifier, dispersant, or foam-forming agent).

[0038] The term "wettable powder" above refers to a fine powder obtained by grinding a water-insoluble pesticide active ingredient with the addition of a mineral extender, such as talc or kaolin, and a surfactant.

[0039] The term "water-soluble agent" above refers to a product prepared by combining a water-soluble pesticide active ingredient with a water-soluble substance, such as sugar or ammonium sulfate, as a bulking agent.

[0040] The term "water-soluble granules" above refers to a preparation formed into granules using a water-soluble active ingredient, such as emamectin, and a water-soluble adjuvant.

[0041] The term "encapsulating agent" above refers to a formulation that physically packages sensitive materials using a protective matrix, etc.

[0042] In one aspect, the composition may be one or more selected from the group consisting of a trunk injection composition, a bio-prescription composition, a fertilizer composition, a nutrient composition, a plant strengthening composition, a plant protection composition, a soil conditioner composition, and a yield enhancer composition.

[0043] The term "trunk injection" above refers to a type of medical treatment for treating tree diseases, which involves inserting a needle or drilling a hole into the trunk of a tree to allow medication to be injected.

[0044] In addition, the above composition may include micronutrients, natural elements, natural oil extracts, etc.

[0045] The above term "micronutrients" refers to elements that are indispensable for plant growth, even in extremely small amounts, such as zinc, iron, manganese, copper, boron, cobalt, vanadium, selenium, silicon, nickel, etc.

[0046] The above term "natural element" means an element that is not newly created by an artificial nuclear reaction, such as technetium, francium, astatine, etc.

[0047] The term "natural extract" above refers to a mixture in which many detailed components are combined, compared to refined single components.

[0049] Another aspect provides a method for controlling fire blight in fruit trees, comprising the step of treating a plant or the soil on which the plant is planted with the above-mentioned control composition.

[0050] The above terms, such as "Erwinia amylovora," "Erwinia pyrifolia," "lysation," "bacteriophage," "control," and "fruit tree fire blight," may fall within the aforementioned scope.

[0051] In one aspect, the fruit tree fire blight may be one or more selected from the group consisting of fire blight and black blight.

[0052] In one aspect, the above plant is Erwinia amylovora ( Erwinia amylovora ) or Erwinia piripoliae( Erwinia pyrifoliae It refers to a plant in which ) is detected, is expected to be detected, or has developed or is expected to develop fire blight or black rot, and may be one or more plants selected from the group consisting of, for example, apple, pear, rose, loquat, quince, apricot, peach, Japanese plum, plum, aronia, cherry, cherry, and rose.

[0053] In one aspect, when the above-mentioned pest control composition is applied to the soil, the composition may exhibit lytic activity against fungi causing disease in plants by being absorbed through the roots of the plant or by being absorbed into the stem or leaves, etc. by evaporation.

[0054] In one aspect, the treatment may involve treating the composition directly or indirectly, for example, by injecting it using an injectable agent, using it diluted with another liquid, or spraying it using a spray.

[0055] The method for controlling fruit tree fire blight according to daily patterns is Erwinia amylovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliea It has excellent lytic activity against ). Effects of the invention

[0057] According to the bacteriophage Fifi011 of the present invention, it has lytic activity (killing ability) against both Erwinia amylovara and Erwinia pyrifolia, so it can effectively deal with fire blight in fruit trees, prevent the emergence of antibiotic-resistant strains, and does not affect the environment, so safe and effective control of fire blight in fruit trees can be expected. Brief explanation of the drawing

[0058] Figure 1 shows the results of confirming the lytic activity of bacteriophage Fifi011 against the causative agent of fruit tree fire blight using plaque. Figure 2 is the result of confirming the morphology of the bacteriophage Fifi011 of the present invention using an electron microscope. Figure 3 is the result of confirming the temperature stability of the bacteriophage Fifi011 of the present invention. Figure 4 is the result of confirming the pH stability of the bacteriophage Fifi011 of the present invention. Figure 5 is a diagram showing the phylogenetic tree of the bacteriophage Fifi011 of the present invention. Figure 6 shows the results of comparing the growth inhibitory effect of the bacteriophage Fifi011 of the present invention on target bacteria at different concentrations (MOI). Specific details for implementing the invention

[0059] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0061] Examples

[0062] Example 1. Isolation of bacteriophage Fifi011 and confirmation of lytic activity

[0063] (1) Pretreatment of environmental samples for bacteriophage isolation

[0064] To isolate bacteriophages, soil and water samples were collected from an orchard (Anseong) affected by fire blight and the surrounding area. 10 g of soil sample and 10 ml of water sample were placed in a 50 ml conical tube, and 10 ml of TSB (tryptic soy broth) was added. To this, Erwinia amylovora (shaken for 24 hours) Erwinia amylovora ) or Erwinia piripoliae( Erwinia pyrifoliea 1 ml of a mixed culture solution, prepared by mixing each of the ) in equal proportions, was added to each sample and incubated with shaking at 27°C for 24 hours. The cultured samples were centrifuged at 10,000×g for 5 minutes, and only the supernatant was transferred to a new tube. The resulting samples were then filtered through a 0.2 μm pore size filter to completely remove bacteria.

[0066] (2) Dotting assay

[0067] A dotting assay was performed to confirm the presence of bacteriophages in various environmental samples. Sterilized media were prepared by adding 0.4% soft agar to TSB and heated to 40°C to 50°C. Erwinia amylovora cultured overnight with shaking in 3 ml of the medium ( Erwinia amylovora ) or Erwinia piripoliae( Erwinia pyrifolieaAdd 15 μl of ) and mix gently to prevent air bubbles, then pour thinly onto TSA (tryptic soy agar) and let it solidify for 10 minutes. Drop 10 μl of the filtered environmental sample onto the medium containing the bacterial strain uniformly, and incubate statically at 27°C. When dropping multiple environmental samples onto a single medium, maintain sufficient spacing to prevent mixing. After 24 hours of incubation, if no bacteria grow on the sample portion and it remains clear or plaque is observed, it is determined to be positive.

[0069] (3) Plaque assay (overlay assay)

[0070] An overlay assay was performed to isolate bacteriophages in purity. A sterile medium was prepared by adding 0.4% soft agar to TSB and heated to 40°C to 50°C. Erwinia amylovora cultured overnight with shaking in 3 ml of the medium ( Erwinia amylovora ) or Erwinia piripoliae( Erwinia pyrifoliea 15 μl of ) and 10 μl of the positive sample in which bacteriophages were confirmed were added and mixed without forming bubbles, then poured thinly into TSA, allowed to solidify for 10 minutes, and incubated statically at 27°C for 24 hours. At this time, the positive sample must be serially diluted at different concentrations to obtain a single plaque. After 24 hours of incubation, plaques of different sizes or shapes were selected, placed in 1 ml of saline-magnesium (SM) buffer, and refrigerated. The same process was repeated at least 5 times for the bacteriophages extracted from each plaque to isolate a single bacteriophage.

[0072] (4) Confirmation of lytic activity of bacteriophage Fifi011

[0073] To confirm the lytic activity of the above-isolated bacteriophage Fifi011, Erwinia Amilovora ( Erwinia amylovora ) and Erwinia Piripoliae ( Erwinia pyrifoliae Plaque was identified using the ) strain.

[0074] As a result, bacteriophage Fifi011 is the causative agent of fruit tree fire blight. Erwinia amylovora and Erwinia pyrifoliae It formed a clear plaque and showed strong lytic activity (Fig. 1).

[0076] Example 2. Morphological analysis of bacteriophage Fifi011

[0077] To promote bacteriophage proliferation, fire blight bacteria were cultured in 3 ml of TSB with shaking for 24 hours. After re-cultured 2 ml of fire blight bacteria, cultured with shaking for 24 hours, in 200 ml of TSB, the OD 600 If it becomes 0.5, it becomes 10 8 1 ml of phage at a concentration of PFU / ml was added, followed by shaking incubation for 24 hours. To remove impurities from the sample after 24 hours of shaking incubation, the sample was centrifuged at 10,000×g for 10 minutes, and the supernatant was filtered through a 0.22 μm pore size filter. The bacteriophage sample was treated with 5 g of Polyethylene Glycol 8000 (PEG 8000) and 10 ml of 5 M NaCl, and then incubated at 4°C for 24 hours. After 24 hours of incubation, the sample was centrifuged at 9,000 g for 20 minutes, the supernatant was removed, and the precipitate was suspended in SM buffer to prepare the sample. 3 ml of CsCl density layers (1.3 g / ml, 1.45 g / ml, 1.5 g / ml, and 1.7 g / ml) were stacked in reverse order in an ultracentrifuge tube, followed by the stacking of the concentrated phage sample on top, and the tube was centrifuged at 785,500×g for 2 hours to obtain purified phages. The purified phages were dialyzed with SM buffer and filtered through a 0.22 μm pore size filter. The filtered phage sample was stained with a 2% uranyl acetate solution, and its morphology was examined using a transmission electron microscope (Fig. 2).

[0078] As a result, bacteriophage Fifi011 Podoviridae It was confirmed that it belongs to the family.

[0080] Example 3. Analysis of the host range of bacteriophage Fifi011

[0081] To confirm the host range of bacteriophage Fifi011, 94 species isolated in Korea Erwinia amylovora and 25 types Erwinia pyrifoliae A dotting assay was used for host range analysis, and the result was determined to be positive if plaque was confirmed after 24 hours of incubation. Specifically, the formation of a clear halo was indicated as + and the absence as -, as shown in Table 1 below. All experiments were performed in 3 to 9 replicates, and the average value of the stage scores of the observed plaques was recorded.

[0082] No. species strain Lytic activity of phage Fifi011 1 Erwinia amylovora YKB 14715 + 2 Erwinia amylovora YKB 14740 + 3 Erwinia amylovora YKB 14742 + 4 Erwinia amylovora YKB 14748 + 5 Erwinia amylovora YKB 14750 + 6 Erwinia amylovora YKB 14754 + 7 Erwinia amylovora YKB 14756 + 8 Erwinia amylovora YKB 14758 + 9 Erwinia amylovora YKB 14768 + 10 Erwinia amylovora YKB 14770 + 11 Erwinia amylovora YKB 14776 + 12 Erwinia amylovora YKB 14778 + 13 Erwinia amylovora YKB 14787 + 14 Erwinia amylovora YKB 14806 + 15 Erwinia amylovora YKB 14808 + 16 Erwinia amylovora YKB 14814 + 17 Erwinia amylovora YKB 14818 + 18 Erwinia amylovora YKB 14820 + 19 Erwinia amylovora YKB 14822 + 20 Erwinia amylovora TS3128 + 21 Erwinia amylovora RA0023 + 22 Erwinia amylovora RA0024 + 23 Erwinia amylovora RA0025 + 24 Erwinia amylovora RA0026 + 25 Erwinia amylovora RA0027 + 26 Erwinia amylovora RA0028 + 27 Erwinia amylovora RA0029 + 28 Erwinia amylovora RA0030 + 29 Erwinia amylovora RA0031 + 30 Erwinia amylovora RA0032 + 31 Erwinia amylovora RA0033 + 32 Erwinia amylovora RA0034 + 33 Erwinia amylovora RA0035 + 34 Erwinia amylovora RA0036 + 35 Erwinia amylovora RA0037 + 36 Erwinia amylovora RA0038 + 37 Erwinia amylovora RA0039 + 38 Erwinia amylovora RA0040 + 39 Erwinia amylovora RA0041 + 40 Erwinia amylovora RA0042 + 41 Erwinia amylovora RA0043 + 42 Erwinia amylovora RA0044 + 43 Erwinia amylovora RA0045 + 44 Erwinia amylovora RA0046 + 45 Erwinia amylovora RA0047 + 46 Erwinia amylovora RA0048 + 47 Erwinia amylovora RA0049 + 48 Erwinia amylovora RA0050 + 49 Erwinia amylovora RA0051 + 50 Erwinia amylovora RA0052 + 51 Erwinia amylovora RA0053 + 52 Erwinia amylovora RA0054 + 53 Erwinia amylovora RA0055 + 54 Erwinia amylovora RA0056 + 55 Erwinia amylovora RA0057 + 56 Erwinia amylovora RA0058 + 57 Erwinia amylovora RA0059 + 58 Erwinia amylovora RA0060 + 59 Erwinia amylovora RA0061 + 60 Erwinia amylovora RA0062 + 61 Erwinia amylovora RA0063 + 62 Erwinia amylovora RA0064 + 63 Erwinia amylovora RA0065 + 64 Erwinia amylovora RA0066 + 65 Erwinia amylovora RA0067 + 66 Erwinia amylovora RA0068 + 67 Erwinia amylovora RA0069 + 68 Erwinia amylovora RA0070 + 69 Erwinia amylovora RA0071 + 70 Erwinia amylovora RA0072 + 71 Erwinia amylovora RA0073 + 72 Erwinia amylovora RA0074 + 73 Erwinia amylovora RA0075 + 74 Erwinia amylovora RA0076 + 75 Erwinia amylovora RA0077 + 76 Erwinia amylovora RA0078 + 77 Erwinia amylovora RA0079 + 78 Erwinia amylovora RA0080 + 79 Erwinia amylovora RA0081 + 80 Erwinia amylovora RA0082 + 81 Erwinia amylovora RA0083 + 82 Erwinia amylovora RA0084 + 83 Erwinia amylovora RA0085 + 84 Erwinia amylovora RA0086 + 85 Erwinia amylovora RA0087 + 86 Erwinia amylovora RA0088 + 87 Erwinia amylovora RA0089 + 88 Erwinia amylovora RA0090 + 89 Erwinia amylovora RA0091 + 90 Erwinia amylovora RA0092 + 91 Erwinia amylovora RA0093 + 92 Erwinia amylovora RA0094 + 93 Erwinia amylovora RA0095 + 94 Erwinia amylovora RA0096 + 95 Erwinia pyrifoliae Ep12327 + 96 Erwinia pyrifoliae RP0098 + 97 Erwinia pyrifoliae RP0099 + 98 Erwinia pyrifoliae RP0100 + 99 Erwinia pyrifoliae RP0101 + 100 Erwinia pyrifoliae RP0102 + 101 Erwinia pyrifoliae RP0103 + 102 Erwinia pyrifoliae RP0104 + 103 Erwinia pyrifoliae RP0105 + 104 Erwinia pyrifoliae RP0318 + 105 Erwinia pyrifoliae RP0107 + 106 Erwinia pyrifoliae RP0108 + 107 Erwinia pyrifoliae RP0109 + 108 Erwinia pyrifoliae RP0110 + 109 Erwinia pyrifoliae RP0111 + 110 Erwinia pyrifoliae RP0112 + 111 Erwinia pyrifoliae RP0113 + 112 Erwinia pyrifoliae RP0114 + 113 Erwinia pyrifoliae RP0115 + 114 Erwinia pyrifoliae RP0116 + 115 Erwinia pyrifoliae RP0117 + 116 Erwinia pyrifoliae RP0118 + 117 Erwinia pyrifoliae RP0119 + 118 Erwinia pyrifoliae RP0120 + 119 Erwinia pyrifoliae RP0121 +

[0084] Looking at Table 1 above, bacteriophage Fifi011 is the causative agent of fruit tree fire blight. Erwinia amylovora and Erwinia pyrifoliea It was confirmed that they exhibited strong lytic activity by forming plaques on all of them.

[0086] Example 4. Check the temperature and pH stability of Fifi011

[0087] For bacteriophage Fifi011, changes in activity at various temperatures and pH levels were confirmed.

[0088] The phage was treated at 30℃, 40℃, and 50℃ for 1 hour, and the change in activity concentration was confirmed through a dotting assay. It was confirmed that the phage exhibited excellent lytic activity against fire blight bacteria even when treated at 40℃ for 1 hour (Fig. 3).

[0089] In addition, the phage was exposed to various pH values ​​from 5 to 10 for 1 hour, and the change in active concentration was confirmed through a dotting assay. As a result, it was confirmed that it exhibited excellent lytic activity against fire blight bacteria in the above pH range (Fig. 4).

[0090] Thus, it can be seen that the bacteriophage Fifi011 of the present invention possesses stable and excellent activity even under poor temperature and pH conditions.

[0092] Example 5. 박하리오파지 Fifi011의 장기서열 오스토통 서이

[0093] For the sequencing of bacteriophage Fifi011, the outer envelope was removed by treating the phage particles with protease, and the phage DNA was extracted using Norgen's phage DNA isolation kit. After constructing a library using the TruSeq Nano DNA kit, next-generation sequencing (NGS) was performed based on the Illumina HiSeqXTen platform. The obtained entire reads were assembled to obtain single contigs.

[0094] The function of each Open reading frame predicted by sequence analysis was analyzed using the Basic Local Alignment Search Tool (BLAST) and HHpred, and the results are shown in Table 2 below.

[0095] [Table 2]

[0096]

[0098] In addition, using the genomic information of the above bacteriophage, a phylogenetic tree was analyzed using Virus Classification and Tree Building Online Resource (VICTOR) to roughly determine where it belongs phylogenetically, and the results are shown in Figure 5.

[0099] The bacteriophage Fifi011 of the present invention was deposited as a microorganism under accession number KACC 97043P.

[0101] Example 6. Evaluation of control concentration of bacteriophage Fifi011

[0102] The growth inhibitory effect of bacteriophage Fifi011 on pathogens was compared according to concentration (Multiplicity of infection, MOI).

[0103] The pathogen is prepared by shaking and culturing it on TSB medium the day before, and then re-cultured after 24 hours. Subsequently, the bacterial concentration is OD600nm=0.5(108 When it reaches CFU / ml, decimal dilute to 10 in TSB medium 5 The inoculation concentration was adjusted to CFU / ml. 180 μl of bacterial culture solution was dispensed into 96-well plates, and 20 μl of phage was inoculated at multiplicity of infection (MOI) of 0.001, 0.1, 10, and 1000. Subsequently, the plates were incubated for 24 hours at 27°C and 120 rpm using a microplate reader, and the bacterial OD values ​​were measured at one-hour intervals.

[0104] As a result, as shown in Figure 6, it was found that the growth inhibition effect of fire blight pathogens was excellent even at low concentrations of phage concentration (MOI) in the range of 0.001 to 0.1.

[0105] Accordingly, according to the present invention, it can be used as a pesticide for controlling fire blight and eggplant black blight, which is environmentally friendly and highly effective compared to chemical pesticides.

[0107] Depository Name: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Accession Number: KACC97043P Date of Deposit: 2023-09-07

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 A composition for controlling fire blight in fruit trees comprising the bacteriophage Fifi011 of accession number KACC 97043P, wherein the composition is treated at a concentration of 0.001 to 0.1 MOI. Claim 8 delete Claim 9 delete Claim 10 A pest control composition according to claim 7, wherein the composition further comprises an antibiotic. Claim 11 A control composition according to claim 10, wherein the antibiotic is streptomycin. Claim 12 A pest control composition according to claim 7, wherein the composition is one or more formulations selected from the group consisting of wettable powders, granular wettable powders, liquid wettable powders, liquids, aqueous solutions, water-soluble granules, and encapsulating agents. Claim 13 A pest control composition according to claim 7, wherein the composition is one or more selected from the group consisting of a trunk injection composition, a biological agent composition, a fertilizer composition, a nutrient composition, a plant strengthening composition, a plant protection composition, a soil conditioner composition, and a yield enhancer composition. Claim 14 A method for controlling fire blight in fruit trees, comprising the step of treating a plant or soil on which the plant is planted with the control composition of claim 7. Claim 15 delete Claim 16 A method according to claim 14, wherein the plant is one or more selected from the group consisting of apple, pear, rose, loquat, quince, apricot, peach, plum, apricot, aronia, cherry, cherry, and rose. Claim 17 A pesticide for controlling fire blight in fruit trees comprising the composition of claim 7. Claim 18 delete

Images