Novel pectobacterium cartovorum-specific bacteriophage and the use thereof

Abstract

The bacteriophage KFS-PCC6 of the present invention has the effect of specific killing and controlling ability against Pectobacterium carotoborum. Specifically, compared to existing chemical antimicrobial substances, the bacteriophage KFS-PCC6 has very high specificity against Pectobacterium carotoborum, excellent lytic activity, the ability to mass-produce by infecting and multiplying target strains, and the advantage of being stable against physicochemical stimuli. In addition, it has excellent acid and heat resistance, as well as stability against ultraviolet rays and agricultural antibiotics, allowing it to be used in combination with other existing antimicrobial substances in various temperature and pH ranges or in actual agricultural environments, making it useful for related industries.

Description

Technology Field

[0001] The present invention relates to a novel bacteriophage KFS-PCC6 having a specific killing ability against Pectobacterium carotoborom, said bacteriophage, and said use. Background Technology

[0002] Pectobacterium carotoborum ( Pectobacterium cartovorum subsp. carotovorum Pectobacterium carotobolum (PCC) is a Gram-negative pathogenic bacterium widely distributed in agricultural soils, primarily found in plants, flowers, or the soil used for cultivation. Pectobacterium carotobolum exists in host plants, their residues, and soil, and invades plants through wounds caused by insects, agricultural tools, or cold damage, causing soft rot in various fresh vegetables, including lettuce (including napa cabbage), potatoes, tomatoes, and onions. Although optimal growth conditions are humid and between 26 and 28 degrees Celsius, it can grow even at 36 degrees, causing serious damage to summer fresh vegetable production, particularly napa cabbage.

[0003] Soft rot, also known as microrot, develops when the cell walls of host plants are broken down and cell membranes are damaged by plant cell wall-degrading enzymes produced by Pectobacterium carotobolum, such as cellulase, protease, pectate lyase, and polygalacturonase. Initially, the disease appears as a wet residue on the plant surface, gradually causing decay symptoms where the tissue becomes mushy accompanied by a distinctive odor. Since Pectobacterium carotobolum survives in the soil for a relatively long time and can occur throughout the crop's entire growth period, and because it spreads rapidly to other tissues once infected, even occurring during storage, the economic losses caused by soft rot are very severe.

[0004] Traditionally, the control of soft rot in crops has primarily involved developing resistant varieties, using copper compounds such as copper hydroxide and copper sulfate, or employing agricultural antibiotics including oxolinic acid, validamycin, streptomycin, kasugamycin, and oxytetracycline. However, variety development is time-consuming and costly, and developed varieties suffer from the disadvantage of losing resistance relatively quickly. Furthermore, chemical agents such as copper compounds and agricultural antibiotics raise issues regarding the emergence of resistant strains, the death of other beneficial microorganisms, and environmental pollution caused by residual toxicity. As a countermeasure, biological control technologies are gaining prominence, and among these, research utilizing bacteriophages that specifically and effectively kill bacteria is receiving particular attention.

[0005] Bacteriophages are viruses that specifically infect bacteria to inhibit and suppress their growth. They possess either a lytic or lysogenic life cycle, and among these, bacteriophages with a lytic life cycle are primarily utilized as biological control agents. Bacteriophages with a lytic life cycle bind to specific sites on host bacteria to infect them; after performing gene replication and assembly within the host, progeny bacteriophages destroy the bacterial cell wall and are released, thereby killing the bacteria. Furthermore, compared to antibiotics, they have the advantages of high host specificity and relative stability against environmental factors such as temperature and pH. Additionally, they can exhibit antimicrobial activity regardless of antibiotic susceptibility, and compared to antibiotics, they require shorter development times and lower development costs. Although bacteriophage products have been approved by the EPA in 2005 and are being used as agricultural pesticides, they are limited to products for controlling bacterial spot, blight, tomato canker, citrus canker, and fire blight in tomatoes, so there is a continuous demand for the development of bacteriophage products for controlling Pectobacterium carotoborum.

[0006] Accordingly, the inventors focused on developing technology for the isolation and purification of bacteriophages and their application, which are effective in controlling only the pathogenic bacteria to be controlled using bacteriophages with excellent specificity and preventing infectious diseases caused by them. As a result, the inventors isolated and purified a bacteriophage with specific killing ability against Pectobacterium carotoborum from the soil of a cabbage field and named it KFS-PCC6. Furthermore, the present invention was completed by confirming the bactericidal and bacteriostatic effects of KFS-PCC6, stability against temperature, pH, and agricultural antibiotics, and morphological characteristics, and by confirming novelty through genetic analysis. The problem to be solved

[0007] The object of the present invention is Pectobacterium carotoborum ( Pectobacterium cartovorum A novel bacteriophage KFS-PCC6 (accession number KCTC14871BP) containing a specific killing ability against ) can be provided.

[0008] In addition, the object of the present invention is a Pectobacterium carotoborum comprising the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP) Pectobacterium cartovorum A composition for killing can be provided.

[0009] In addition, the objective of the present invention is to provide an antimicrobial composition for inhibiting the causative agent of vegetable soft rot, comprising the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP).

[0010] In addition, the object of the present invention is to provide a composition for adjuvant antibiotics for agriculture comprising the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP).

[0011] In addition, the objective of the present invention is to provide a food detergent or disinfectant comprising an antimicrobial composition for inhibiting the causative agent of vegetable soft rot. means of solving the problem

[0012] Accordingly, to achieve the above objective, the present invention relates to Pectobacterium carotoborum ( Pectobacterium cartovorum Provides a novel bacteriophage KFS-PCC6 (accession number KCTC14871BP) that includes a specific killing ability against ).

[0013] In addition, the present invention relates to Pectobacterium carotoborum (including the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP) Pectobacterium cartovorum ) Provides a composition for killing.

[0014] In addition, the present invention provides an antimicrobial composition for inhibiting the causative agent of vegetable soft rot, comprising the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP).

[0015] In addition, the present invention provides a composition for adjuvant antibiotics for agriculture comprising the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP).

[0016] In addition, the present invention provides a food detergent or disinfectant comprising an antimicrobial composition for inhibiting the causative agent of vegetable soft rot. Effects of the invention

[0017] The bacteriophage KFS-PCC6 of the present invention has the effect of specific killing and controlling ability against Pectobacterium carotoborum. Specifically, compared to existing chemical antimicrobial substances, the bacteriophage KFS-PCC6 has very high specificity against Pectobacterium carotoborum, excellent lytic activity, the ability to mass-produce by infecting and multiplying target strains, and the advantage of being stable against physicochemical stimuli. In addition, it has excellent acid and heat resistance, as well as stability against ultraviolet rays and agricultural antibiotics, allowing it to be used in combination with other existing antimicrobial substances in various temperature and pH ranges or in actual agricultural environments, making it useful for related industries. Brief explanation of the drawing

[0018] Figure 1 is a figure confirming the lytic plaque-forming activity of bacteriophage KFS-PCC6 against Pectobacterium carotoborum KACC 14893. Figure 2 is a figure confirming the morphological characteristics of bacteriophage KFS-PCC6 through a transmission electron microscope. Figure 3 is a figure confirming the temperature stability of bacteriophage KFS-PCC6. Figure 4 is a figure confirming the results of examining the pH stability of bacteriophage KFS-PCC6. Figure 5 is a figure confirming the stability of bacteriophage KFS-PCC6 against UV exposure. Figure 6 is a figure confirming the stability of bacteriophage KFS-PCC6 against agrobiotic antibiotics. Figure 7 shows the results of a challenge assay examining the inhibitory activity of bacteriophage KFS-PCC6 against Pectobacterium carotoborum. Specific details for implementing the invention

[0019] 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.

[0020] The bacteriophage KFS-PCC6 of the present invention is a bacteriophage isolated and purified from a sample collected from the soil of a tomato field at a farm in Daegu, and was named bacteriophage KFS-PCC6 and deposited at the National Center for Biological Resources on February 23, 2022, under accession number KCTC 14871BP.

[0021] The present invention relates to an indicator strain of Pectobacterium carotoborum subspecies carotoborum ( Pectobacterium cartovorum subsp. carotovorum Provides bacteriophage KFS-PCC6 (accession number KCTC 14871BP), which has specific killing ability against KACC 14893 (PCC).

[0022] In the present invention, the term "bacteriophage" refers to a bacterium-specific virus that infects a specific bacterium and inhibits and suppresses the growth of said bacterium, and means a virus comprising single or double-stranded DNA or RNA as genetic material.

[0023] The bacteriophage KFS-PCC6 of the present invention exhibits excellent lytic activity against the host strain Pectobacterium carotoborum KACC 14893, and the bacteriophage KFS-PCC6 has a myovirium, which is a form having an angular head and a contractile tail ( Myoviridae It was confirmed that it possesses the characteristics of ). In addition, the bacteriophage KFS-PCC6 has lytic activity specifically limited to the host strain Pectobacterium carotoborum and has excellent stability against heat, pH, and agricultural antibiotics.

[0024] The bacteriophage KFS-PCC6 of the present invention maintains lytic activity within the range of -20°C to 50°C, but is not limited thereto.

[0025] The bacteriophage KFS-PCC6 of the present invention maintains lytic activity within the range of pH 3.0 to pH 11.0, but is not limited thereto.

[0026] The bacteriophage KFS-PCC6 of the present invention maintains lytic activity after irradiation with ultraviolet light at a wavelength of 365 nm, but is not limited thereto.

[0027] In addition, the present invention provides an antibiotic composition comprising the bacteriophage KFS-PCC6.

[0028] The bacteriophage KFS-PCC6 of the present invention maintains lytic activity even in a suspension of five types of agricultural antibiotics including oxolinic acid, streptomycin, validamycin, oxytetracycline, and kasugamycin, but is not limited thereto.

[0029] In the present invention, the term "antibiotic composition" refers to a preparation that can kill bacteria when provided to plants or crops in the form of a drug, and is a collective term for preservatives, fungicides, antibiotics, and antimicrobial agents.

[0030] The bacteriophage KFS-PCC6 of the present invention has high specificity against infectious diseases of Pectobacterium carotoborum compared to existing antibiotics, so it can kill only specific pathogens without killing beneficial bacteria and does not induce drug resistance or resistance, so it can be used as a novel alternative antibiotic with a longer life cycle compared to existing antibiotics.

[0031] The bacteriophage of the present invention is characterized by having specific proliferation inhibition or killing ability against Pectobacterium carotoborum. More specifically, it has antimicrobial activity against Pectobacterium carotoborum, but is not limited thereto; however, in the embodiments of the present invention, it was confirmed that there is lytic activity specifically against Pectobacterium carotoborum among plant pathogenic bacteria. The Pectobacterium carotoborum is characterized as a pathogenic bacterium that, upon infection, causes diseases such as soft rot in vegetables. That is, the bacteriophage of the present invention is characterized by having antimicrobial activity against Pectobacterium carotoborum.

[0032] In addition, the present invention relates to Pectobacterium carotoborum (including the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP) Pectobacterium cartovorum ) Provides a composition for killing.

[0033] In addition, the present invention provides an antimicrobial composition for inhibiting the causative agent of vegetable soft rot, comprising the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP).

[0034] In addition, the present invention provides a composition for adjuvant antibiotics for agriculture comprising the novel bacteriophage KFS-PCC6 (accession number KCTC14871BP).

[0035] In addition, the present invention provides a food detergent or disinfectant comprising an antimicrobial composition for inhibiting the causative agent of vegetable soft rot.

[0036] Agricultural antibiotics used in agricultural environments are used for the purpose of preventing various diseases. However, the administration of antibiotics for preventive purposes has become problematic because it increases the possibility of antibiotic resistance developing and antibiotics remaining in plants and the agricultural environment can be transferred to humans. Furthermore, if antibiotics are absorbed into the human body through food consumption, they can induce antibiotic resistance, potentially leading to the spread of disease. Additionally, agricultural antibiotics have a broad range of action and do not clearly target specific strains, which increases the probability of multidrug-resistant bacteria developing. Therefore, the bacteriophage KFS-PCC6 of the present invention can be utilized as a new antibiotic that is environmentally friendly and can solve the problems arising from the use of conventional antibiotics.

[0037] In addition, the antibiotic composition or soft rot control composition provided in the present invention may be prepared by separately manufacturing the bacteriophage in the form of an additive and mixing it with other antibiotic substances, or by directly adding it during the manufacture of antibiotic substances. The bacteriophage of the present invention may be in a liquid or dry state, and drying methods may include air drying, natural drying, spray drying, and freeze-drying, but are not limited thereto. Furthermore, the feed may additionally include conventional additives capable of enhancing preservation or stability in addition to the bacteriophage of the present invention.

[0038] In addition, the present invention provides a disinfectant comprising the bacteriophage KFS-PCC6. The disinfectant comprising the bacteriophage KFS-PCC6 of the present invention, which has the ability to kill Pectobacterium carotoborum, can be usefully used as a disinfectant to prevent infection in agricultural environments or during cultivation, storage, distribution, or processing, and can be used as a general household disinfectant, a disinfectant for food and cooking areas and facilities, and for disinfecting various agricultural supplies such as agricultural machinery, drinking water, bedding, egg trays, and transport vehicles.

[0039] In addition, the present invention provides a cleaning agent comprising the bacteriophage KFS-PCC6. Since the bacteriophage KFS-PCC6 of the present invention has a specific killing ability against Pectobacterium carotoborum, it can also be used to clean the surface of crops that have been exposed to or are likely to be exposed to said strain.

[0040] The antibiotic composition, vegetable soft rot control composition, disinfectant, or detergent of the present invention comprising the bacteriophage KFS-PCC6 is 10 3 -10 10 Contains bacteriophages of PFU / ml, preferably 10 6 -10 9 It contains bacteriophages in PFU / ml. The term "PFU (plaque forming unit)" as used in this invention is a unit that quantifies the lytic plaques that appear after bacterial death caused by bacteriophages.

[0041] In addition, any one selected from the antibiotic composition, the composition for controlling vegetable soft rot, the disinfectant, or the detergent of the present invention may further include a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the administered compound for the purpose of stable formulation suitable for use. As for the pharmaceutical carriers acceptable for the composition formulated as a liquid solution, they may be sterile and biocompatible, and may include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, extenders, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0042] Any one selected from the antibiotic composition, vegetable soft rot control composition, disinfectant, or detergent of the present invention can be used by applying or spraying it onto a site where the disease appears.

[0043] Suitable application, spraying, and dosage amounts of any one selected from the antibiotic composition, vegetable soft rot control composition, disinfectant, or cleaning agent of the present invention vary depending on factors such as the formulation method, the method of administration, the severity of disease symptoms of the target crop, the administration time, the administration route, and the sensitivity to response, and will be easily understood by those skilled in the art.

[0044] As an example of the above surfactant, alkyl (C 8- C 12 )arylsulfonate, dialkyl(C 8- C 12 )arylsulfonate, dialkyl(C 8- C 12 )sulfosuccinate, ligninsulfonate, naphthalenesulfonate condensate, alkyl(C8- C 12 )Naphthalenesulfonate formalin condensate, polyoxyethylenealkyl(C 8- C 12 Sodium or calcium salts of sulfonate compounds such as phenylsulfonate, sodium or calcium salts of succinate compounds such as polyoxyalkylene succinate, anionic surfactants such as sodium benzoate, polyoxy(C 12- C 18 )ether, polyoxyethylenealkyl(C 8- C 12 It will be readily understood by those skilled in the art that one or more selected from the group consisting of nonionic surfactants such as phenyl polymers, polycarboxylates, Triton 100, and Tween 80 may be used in combination, and that other surfactants other than these may also be used.

[0045] As fillers, bentonite, talc, clay, kaolin, calcium carbonate, silica sand, pumice, diatomaceous earth, acid clay, zeolite, perlite, ammonium sulfate, glucose, dextrin, soybean flour, rice, wheat, red clay, glucose and starch, water, etc. may be used alone or in a mixture of two or more types, and it will be easily understood by a person with ordinary knowledge in the relevant technical field that other fillers other than these may also be used.

[0046] Terms not otherwise defined in this specification have the meanings commonly used in the technical field to which the present invention belongs.

[0047] The present invention will be explained in detail below through examples and formulation examples. However, the following examples and formulation examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and formulation examples.

[0049] <Example 1> Isolation and Culture of Novel Bacteriophages

[0050] <Example 1-1> Screening of bacteriophages infecting Pectobacterium carotoborum and isolation of single bacteriophages

[0051] In the sample, Pectobacterium carotoborum subspecies carotoborum ( Pectobacterium cartovorum subsp. carotovorum To analyze the presence or absence of bacteriophages specific to PCC, a soft agar overlay method was performed. The soft agar overlay method involves mixing top-agar (0.4% agar) medium with a host strain (PCC), dispensing and culturing it on a solid medium, and then observing the lytic ability of the bacteriophage through the formation of lysis spots.

[0052] To isolate bacteriophages, soil from a tomato field collected from a farm in Daegu Metropolitan City was used as a sample. Specifically, 25 g of the sample and the shaken culture medium (OD) of the host strain (PCC KACC 14893) were added to 225 ml of LB medium (Luria-Bertani, Difco Inc., USA). 640 = 0.4) 1% (v / v) was mixed and incubated at 28°C at 160 rpm for 24 hours. The culture medium was centrifuged at 6,000 rpm for 20 minutes, and the supernatant was filtered through a 0.25 μm filter. Subsequently, 4 ml of TA soft agar (0.4% (w / v) agar, nutrient broth, NaCl, MgSO4, CaCl2) and PCC KACC 14893 shaken culture medium (OD 640 = 0.4) After mixing 200 μl, the mixture was dispensed onto LB agar plates (1.5% (w / v) agar, Luria-Bertani) and dried by leaving it at room temperature for 10 minutes. 10 μl of the serially diluted solution of the sample was then spotted onto the plates, and the presence of bacteriophages was confirmed by observing the formation of a clear zone after incubation at 28°C for 24 hours. The presence of bacteriophages was confirmed through the formation of a clear zone, and the bacteriophages were identified as Pectobacterium carotoborum subspecies carotoborum ( Pectobacterium cartovorum subsp. carotovorumIt was confirmed that it is a bacteriophage with lytic activity specific to KACC 14893 (PCC).

[0053] After confirming the presence of bacteriophages, a single bacteriophage was isolated from the sample using a plaque assay. 4 ml of soft agar (0.4% agar) medium, 200 μl of a shaken culture of Pectobacterium carotoborum KACC14893, and 100 μl of the sample solution in which the presence of the bacteriophage was confirmed were mixed and dispensed onto a solid LB plate medium. The mixture was then incubated at 28°C for 24 hours to confirm plaque formation. Only one of the formed plaques was recovered using a sterile tip, mixed with 900 μl of SM buffer solution (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4), and incubated at room temperature for 1 hour to obtain a bacteriophage solution. Plaque analysis was performed again using the bacteriophage solution obtained above, and this process was repeated at least 10 times until the size and shape of the formed plaques were all similar to isolate a single bacteriophage. The results confirming the formation of the plaque of the isolated single bacteriophage are shown in Figure 1.

[0055] <Example 1-2> Mass Culture and Purification of Bacteriophages

[0056] In order to purify the bacteriophage obtained in Example 1-1 above, the bacteriophage was cultured and proliferated in large quantities using the host bacterium Pectobacterium carotobolum KACC 14893, and the bacteriophage was isolated and purified therefrom.

[0057] Specifically, Pectobacterium carotoborum KACC 14893 was cultured at 28°C for 16 hours, and 1% (v / v) of the strain culture was added to 3 ml of TA liquid medium (nutrient broth, NaCl, MgSO4, CaCl2) and cultured with shaking at 190 rpm at 28°C for 2 hours and 30 minutes. Then, 1 ml of the single bacteriophage obtained in Example 1.1 was added to the culture medium that had been cultured with shaking, and cultured again at 190 rpm at 28°C for 2 hours and 30 minutes. After culture, the supernatant obtained by centrifugation at 6000 rpm for 20 minutes was filtered using a 0.22 μm filter to isolate only the bacteriophage. In the same way, the amount of TA liquid medium was increased from 3 ml to 8, 20, 200, and 800 ml in sequence, and the bacteriophage concentration (titer) was 10 10 The bacteriophages were expanded until PFU / ml was reached. 10% (w / v) polyethylene glycol (PEG) and 1 M NaCl were added to the mass-produced bacteriophages and incubated at 4°C for 16 hours, after which the mixture was centrifuged at 8,000 rpm for 20 minutes at 4°C. The supernatant was removed, and the remaining pellet was obtained using 6 ml of SM buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4).

[0058] The obtained phages were subjected to ultra-high-speed centrifugation at 22,000 rpm for 2 hours at 4°C after a CsCl density gradient (1.20, 1.30, 1.40, 1.45, 1.50, 1.70 g / ml), and after dialyzing with SM buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4), were stored at 4°C until use.

[0059] The bacteriophage isolated and purified in this manner was named "Bacteriophage KFS-PCC6" and deposited at the Korean Collection for Type Cultures (Korea) on February 23, 2022, and assigned accession number KCTC 14871BP.

[0061] <Example 2> Morphological characteristics of bacteriophage KFS-PCC6

[0062] To characterize the morphological properties of bacteriophage KFS-PCC6, observations were made using a transmission electron microscope (TEM). A copper grid was dipped into 1 ml of bacteriophage KFS-PCC6 for 1 minute, negatively stained with 2% uranyl acetate, and then dried. The morphological characteristics of the phage were observed using an electron microscope H-7100 (Hitachi) at a voltage of 100 kV. The transmission electron microscope observations were performed at the Joint Laboratory of Kyungpook National University, and the results are shown in Figure 2.

[0063] As a result, bacteriophage KFS-PCC6 was confirmed to have a head with a length of 81.09 ± 5.46 nm and a contractile tail with a length ranging from 11.31 ± 2.57 to 174.66 ± 30.2 nm. Through this, the myovirium band having an icosahedral head and a contractile tail ( Myoviridae It was confirmed to have the morphological characteristics of ) (Fig. 2).

[0065] <Example 3> Investigation of Temperature Stability of Bacteriophage KFS-PCC6

[0066] To confirm the temperature stability of bacteriophage KFS-PCC6, the lytic activity (potency) of KFS-PCC6 was measured using a plaque assay at various temperature ranges (-70℃, -20℃, 4℃, 22℃, 37℃, 50℃, 60℃, 70℃, 80℃).

[0067] Specifically, 100 μl of a bacteriophage KFS-PCC6 solution at a concentration of 8 log PFU / ml and 900 μl of SM buffer solution were mixed, and then each was incubated at room temperature for 1 hour at various temperatures (-70℃, -20℃, 4℃, 22℃, 37℃, 50℃, 60℃, 70℃, 80℃). The titer of bacteriophage KFS-PCC6 was measured by plaque assay after reacting at various temperatures for 1 hour, and the results are shown in Figure 3.

[0068] As shown in Figure 3, it was found that the bacteriophage KFS-PCC6 maintained stable activity from -20 to 50°C. It was also confirmed that lytic activity was maintained at 66.7% and 40.0% or higher at -70°C and 60°C, respectively.

[0069] Therefore, it was confirmed that the bacteriophage KFS-PCC6 of the present invention is a bacteriophage with excellent temperature stability.

[0071] <Example 4> Investigation of pH stability of bacteriophage KFS-PCC6

[0072] To confirm the pH stability of bacteriophage KFS-PCC6, 900 μl of SM buffer solution adjusted to various pH ranges (pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) was mixed with 100 μl of bacteriophage KFS-PCC6 at a concentration of 8 log PFU / ml, and then incubated at room temperature for 1 hour. The titer of bacteriophage KFS-PCC6 was measured using a plaque assay after reacting for 1 hour, and the results are shown in Figure 4.

[0073] As shown in Figure 4, it was confirmed that the activity of bacteriophage KFS-PCC6 is stably maintained in the pH range of 3.0 to 11.0. Therefore, it was confirmed that the bacteriophage KFS-PCC6 of the present invention is a bacteriophage with excellent pH stability.

[0075] <Example 5> UV Stability Investigation of Bacteriophage KFS-PCC6

[0076] To confirm the UV stability of bacteriophage KFS-PCC6, KFS-PCC6 was exposed to UV A (365 nm), and its lytic activity (potency) was measured using a plaque assay.

[0077] Specifically, 100 μl of bacteriophage KFS-PCC6 solution at a concentration of 8 log PFU / ml was mixed with 900 μl of SM buffer and dispensed into a 6-well plate, after which a UV lamp (365 nm) was used at a distance of 15 cm at 3500 mW / m² 2 After exposure to UV A wavelength (365 nm) of high intensity, the samples were cultured at room temperature for 1 hour. Subsequently, the titers of the bacteriophage KFS-PCC6 exposed to UV light were measured by plaque assay, and the results are shown in Figure 5.

[0078] As shown in Figure 5, it was found that the bacteriophage KFS-PCC6 maintained over 98% lytic activity even after UV A wavelength treatment. Therefore, it was confirmed that the bacteriophage KFS-PCC6 of the present invention is a bacteriophage with excellent UV stability.

[0080] <Example 6> Investigation of the safety of bacteriophage KFS-PCC6 against agronomic antibiotics

[0081] To confirm the antibiotic stability of bacteriophage KFS-PCC6, the stability of KFS-PCC6 was measured against six types of antibiotics currently approved for use in Korea (streptomycin, oxolinic acid, validamycin A, kasugamycin, polyoxin B, and oxytetracycline).

[0082] Specifically, 1.0 × 10 71 mL of bacteriophage KFS-PCC6 solution at a PFU / mL concentration was mixed with TSB medium solution and incubated for 1 hour in each agricultural antibiotic. At this time, the concentration of the agricultural antibiotic was set to reflect the actual concentration currently used for crops such as cabbage and lettuce. After reacting for 1 hour, the KFS-PCC6 was cultured at 28°C for 18 hours using the soft agar overlay method (plaque assay), and the titer of the remaining bacteriophage KFS-PCC6 was measured. The results are shown in Figure 6.

[0083] As shown in Figure 6, it was confirmed that bacteriophage KFS-PCC6 maintained stability in streptomycin, oxolinic acid, validamycin A, kasugamycin, and oxytetracycline.

[0084] Therefore, since it was confirmed that the bacteriophage KFS-PCC6 of the present invention maintains its activity in various agricultural antibiotics, it was confirmed that it can be applied to combined treatment with agricultural antibiotics when used for the control of vegetable soft rot.

[0086] <Example 7> Lytic effect of bacteriophage KFS-PCC6 on Pectobacterium carotoborum

[0087] A culture of Pectobacterium carotoborum KACC 14893 was inoculated into 100 ml of LB at 1% (v / v), and when the absorbance value of the culture reached approximately 0.4 at 640 nm (10 8 The culture was incubated at 190 rpm at 28°C until the concentration reached CFU / ml. Subsequently, KFS-PCC6 and the bacterial culture medium were mixed and adjusted to MOI (multiplicity of infection) values ​​of 0.01, 0.1, 1.0, and 10, and cultured at 200 rpm at 28°C for 24 hours. A culture of Pectobacterium carotoborum KACC 14893 with SM buffer added was used as a control instead of KFS-PCC6. The initial bacterial concentration was set to 6 Log CFU / mL after dilution, and viable cell counts were measured at 2-hour intervals; the results are shown in Figure 7.

[0088] As a result, the absorbance was significantly lower compared to the control group at all MOIs, and it was confirmed that the lytic activity of KFS-PCC6 persisted for 24 hours. In particular, since the lytic activity of KFS-PCC6 remained high for 24 hours even at the lowest MOI of 0.01, it was confirmed that effective inhibition of Pectobacterium carotoborum is possible with a small amount of phage, thereby confirming the efficient killing ability of KFS-PCC6.

[0090] <Example 8> Whole-genome analysis of bacteriophage KFS-PCC6

[0091] To analyze the whole genome of the bacteriophage KFS-PCC6 purified in Example 1 above, DNA of KFS-PCC6 was extracted using NORGEN’s phage DNA Isolation Kit. The gDNA of KFS-PCC6 was sequenced using the Illumina platform, and the open reading frames (ORFs) of the sequence were annotated with functional groups using BLASTP and RAST databases.

[0092] Genetic analysis revealed that KFS-PCC6 possesses double-stranded DNA, has a total genome size of 44,990 bp (Sequence No. 1), and a GC content of 50.94%. Additionally, a comparative genome analysis with bacteriophages registered in the database using BLAST (http: / / www.ncbi.nih.gov / BLAST) yielded the results shown in Table 1 below. Pectobacterium phage CX5(MN270887.1) and 95.58%, Pectobacterium Although it had the highest identity %) with phage CX5 (MN270888.1), all showed less than 96% identity, so the novelty of KFS-PCC6 was proven.

[0093] The complete gene sequence of bacteriophage KFS-PCC6 is shown as sequence number 1.

[0094] bacteriophage Accession number Homology (Identity %) Pectobacterium phage CX5 MN270887.1 95.58 Pectobacterium phage CX5-1 MN270888.1 95.57 Pectobacterium phage MA2 MN271656.1 95.12 Pseudomonas phage phiNV3 NC047933.1 92.00 Pectobacterium phage PP16 NC031068.2 90.72

[0096] <Example 9> Investigation of Host Specificity of Bacteriophage KFS-PCC6

[0097] To determine whether the above bacteriophage exhibits lytic activity against other plant pathogenic bacteria or food poisoning bacteria other than Pectobacterium carotoborum, the host specificity of KFS-PCC6 was examined.

[0098] After culturing 22 species, including the host strain Pectobacterium carotoborum KACC 14893, plant pathogenic bacteria, and food poisoning bacteria, the formation of lysate was checked using the soft agar overlay method, and the results are shown in Table 2.

[0099] Strain name Whether lytic plaques are formed Pectobacterium carotovorum KACC 14893 O Clavibacter michiganensis subsp. michiganensis KACC 16995 X C . michiganensis subsp. michiganensis KACC 17003 X C . michiganensis subsp. michiganensis KACC 18448 X Pseudomonas syringae pv. actinidiae KACC 10582 X P . syringae pv. actinidiae KACC 10594 X Xanthomonas arboricola pv. plum trees KACC 19950 X X. arboricola pv. plum trees KACC 19951 X X. arboricola pv. plum trees KACC 19952 X X. arboricola pv. plum trees KACC 19953 X Escherichia coli O157:H7 ATCC 10536 X E. coli O157:H7 ATCC 43895 X Bacillus cereus ATCC 14579 X Listeria monocytogenes ATCC 7644 X Salmonella Enteritidis ATCC 13076 X S. Typhimurium ATCC 13311 X S. Dublin NCCP 13700 X S. Heidelberg NCCP 13698 X S. Montevideo NCCP 13704 X S. Newport NCCP 13686 X S. Panama NCCP13694 X Shigella sonnei ATCC 9290 X

[0101] As shown in Table 2 above, it was confirmed that bacteriophage KFS-PCC6 has a narrow range of specificity, as it exhibits lytic activity specifically against Pectobacterium carotoborum. It did not show lytic activity against the other 22 strains.

[0102] From the above experiment, the bacteriophage KFS-PCC6 of the present invention can specifically inhibit only Pectobacterium carotobolum, so it has the advantage of solving the problems of antibiotic-resistant bacteria caused by the misuse of antibiotics and the problem of antibiotic residues in food. Therefore, the bacteriophage KFS-PCC6 of the present invention can effectively prevent Pectobacterium carotobolum infection in agricultural environments.

[0103] As described above, although the present invention has been explained as a preferred embodiment mentioned above, those skilled in the art will be able to readily propose other inventions that are inferior or other embodiments included within the scope of the inventive concept by adding, changing, or deleting other components within the same technical scope. Therefore, the embodiments described above are illustrative in all respects and do not limit the scope of the present invention.

[0104] The following describes examples of pharmaceutical compositions for the prevention of infectious diseases comprising the bacteriophage KFS-PCC6 of the present invention; however, these examples are intended to illustrate examples of formulations that can be prepared according to the present invention, and the scope of the present invention is not limited to these examples.

[0106] <제제예 1>

[0107] Preparation of antibiotic compositions

[0109] <제제예 1-1>

[0110] Manufacturing of injectables

[0111] Bacteriophage KFS-PCC6 1 × 10 6 PFU / ml

[0112] Appropriate amount of sterile distilled water for injection

[0113] Appropriate amount of pH adjuster

[0114] It was prepared according to the conventional method of preparing injectables with the above ingredient content per 1 ampoule (2 mL).

[0116] <제제예 1-2>

[0117] Preparation of liquid formulations

[0118] Bacteriophage KFS-PCC6 1 × 10 6 PFU / ml

[0119] 20 g sugar

[0120] 20 g isomerized sugar

[0121] Appropriate amount of lemon flavor

[0122] Purified water was added to make the total volume 1,000 ml, and the above ingredients were mixed according to the standard method for preparing liquid preparations, then filled into a brown bottle and sterilized to prepare the liquid preparation.

[0124] <제제예 1-3>

[0125] Preparation of tablets

[0126] Bacteriophage KFS-PCC6 1 × 10 6 PFU / ml

[0127] 100 mg of corn starch

[0128] 100 mg lactose

[0129] Magnesium stearate 2 mg

[0130] After mixing the above ingredients, tablets were manufactured by compressing them according to the conventional method of manufacturing tablets.

[0132] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC14871BP Date of Deposit: 2022-02-23

Claims

Claim 1 Pectobacterium carotoborum ( Pectobacterium cartovorum Bacteriophage KFS-PCC6 (accession number KCTC14871BP), containing a specific killing ability against ). Claim 2 Bacteriophage KFS-PCC6 (accession number KCTC14871BP), wherein the bacteriophage of claim 1 is characterized by having lytic activity in the range of -20℃ to 50℃. Claim 3 Bacteriophage KFS-PCC6 (accession number KCTC14871BP), characterized in that, in claim 1, the bacteriophage has lytic activity in the range of pH 3.0 to pH 11.

0. Claim 4 Bacteriophage KFS-PCC6 (accession number KCTC14871BP), wherein the bacteriophage has stable lytic activity at an ultraviolet wavelength of 365 nm in claim 1. Claim 5 In claim 1, the bacteriophage KFS-PCC6 (accession number KCTC14871BP), which exhibits stable lytic activity when used in combination with an antibiotic. Claim 6 In claim 5, the antibiotic is one or more agronomic antibiotics selected from the group consisting of streptomycin, oxolinic acid, validamycin A, kasugamycin, and oxytetracycline, bacteriophage KFS-PCC6 (accession number KCTC14871BP). Claim 7 Pectobacterium carotoborum (including the bacteriophage KFS-PCC6 of claim 1 (accession number KCTC14871BP)) Pectobacterium cartovorum ) Composition for killing. Claim 8 An antimicrobial composition for inhibiting the causative agent of vegetable soft rot, comprising the bacteriophage KFS-PCC6 (accession number KCTC14871BP) of claim 1. Claim 9 A composition for adjuvating antibiotics for agriculture, comprising the bacteriophage KFS-PCC6 (accession number KCTC14871BP) of claim 1. Claim 10 A food detergent comprising an antimicrobial composition for inhibiting the causative agent of vegetable soft rot according to claim 8. Claim 11 A disinfectant comprising an antimicrobial composition for inhibiting the causative agent of vegetable soft rot according to claim 8.

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