BACTERIOPHAGE phiEaSP3 AND USE THEREOF

Abstract

The present invention relates to a novel bacteriophage phiEaSP3 and its uses, and more specifically, to a novel bacteriophage phiEaSP3 (KACC 87007BP) that exhibits specific activity against fruit tree fire blight bacteria and maintains activity even at low temperatures, and a composition for inhibiting or killing fire blight bacteria containing the same.

Description

Technology Field

[0001] The present invention relates to a novel bacteriophage phiEaSP3 and its uses. Background Technology

[0003] Fire blight is a fatal infectious disease affecting apple, pear, and Rosaceae plants, caused by the bacterium *Erwinia amylovora*. The disease manifests as leaves, flowers, branches, stems, and fruits turning black and drying out, resembling burns. The bacteria can be transmitted by insects or wind, and the disease is highly contagious as it invades the host plant through flowers, nectaries, stomata, and wounds. Due to its rapid spread and classification as a prohibited pest under the Plant Quarantine Act, an outbreak requires a three-year ban on planting host plants in the same area, the burial of all host plants in surrounding regions, and the closure of the orchard, resulting in severe economic losses.

[0004] To date, the control of fire blight in fruit trees has primarily been carried out using chemical agents such as copper compounds and antibiotics. However, such control methods have limitations as copper and antibiotic-resistant fire blight strains resulting from the misuse of these chemical agents have already been discovered abroad. As a countermeasure, methods utilizing bacteriophages are being researched; since phages do not affect eukaryotic cells, they are not harmful to humans or animals and can serve as a biological control technology that does not impact environmental components.

[0005] The inventors completed the present invention by discovering that a novel bacteriophage phiEaSP3 isolated from an area where fire blight occurred in fruit trees has specific lytic activity against fire blight pathogens and can be used as a biological control agent. Prior art literature

[0007] Korean Registered Patent No. 10-2025403 The problem to be solved

[0008] The present invention aims to provide a novel bacteriophage having lytic activity specific to fruit tree fire blight pathogens.

[0009] The present invention aims to provide a composition for inhibiting the proliferation or killing fire blight bacteria comprising a bacteriophage.

[0010] The present invention aims to provide a method for controlling fruit trees comprising the step of treating with a composition for inhibiting the proliferation or killing fire blight bacteria containing a bacteriophage. means of solving the problem

[0012] 1. Bacteriophage phiEaSP3 (KACC 87007BP) having lytic activity specific to fruit tree fire blight pathogens.

[0013] 2. In the above 1, a bacteriophage phiEaSP3 having the genome sequence of SEQ ID NO. 1.

[0014] 3. In the above 1, bacteriophage phiEaSP3 having activity at 0℃ to 50℃.

[0015] 4. The bacteriophage phiEaSP3 of 1 above, having activity at pH 3 to 11.

[0016] 5. A composition for inhibiting the proliferation or killing fire blight bacteria comprising any one of the bacteriophages phiEaSP3 (KACC 87007BP) of 1 to 4 above.

[0017] 6. A method for controlling fruit trees comprising the step of treating with the composition for inhibiting the proliferation or killing of fire blight bacteria of 5 above.

[0018] 7. A method for controlling fruit trees according to 6 above, wherein the fruit tree is selected from the group consisting of apple, pear, quince and apricot. Effects of the invention

[0020] The bacteriophage phiEaSP3 of the present invention exhibits specific lytic activity against fruit tree fire blight pathogens. Since the bacteriophage phiEaSP3 of the present invention does not infect Erwinia pyrifoliae, which causes black-shoot blight exhibiting symptoms similar to fruit tree fire blight, it can reduce misdiagnosis of plant diseases or unnecessary control measures.

[0021] The bacteriophage phiEaSP3 of the present invention has a wide active temperature and pH range.

[0022] The bacteriophage phiEaSP3 of the present invention can be used as an eco-friendly biological control agent to replace chemical agents.

[0023] The bacteriophage phiEaSP3 of the present invention has a low likelihood of the emergence of resistant bacteria and high environmental safety. Brief explanation of the drawing

[0025] Figure 1 shows the formation of plaques by bacteriophage phiEaSP3 in an experiment using the fire blight pathogen EaTS3128 as a host. Figure 2 shows the bacteriophage phiEaSP3 of the present invention confirmed by a transmission electron microscope. Figure 3 relates to the stability of phiEaSP3 under environmental factors of temperature, pH, soil, and ultraviolet radiation. Figure 4 confirms the preventive effect of bacteriophage phiEaSP3 against fruit tree fire blight in apple trees. Specific details for implementing the invention

[0026] The present invention provides a novel bacteriophage phiEaSP3 that exhibits specific activity against fruit tree fire blight pathogens, and a composition for inhibiting the proliferation or killing fire blight pathogens containing the same.

[0027] Bacteriophage phiEaSP3 was isolated from soil in Chungju, Chungcheongbuk-do, where fire blight occurred. Bacteriophage phiEaSP3 was deposited with the Korea Agricultural and Biotechnological Research Institute (KACC) on July 13, 2023 (Accession No. KACC 87007BP).

[0028] Bacteriophage phiEaSP3 uses the fire blight bacterium (Erwinia amylovora) as a host. Bacteriophage phiEaSP3 lyses only the fire blight bacterium. It does not lyse the black twig blight bacterium (Erwinia pyrifoliae), which causes similar symptoms.

[0029] The bacteriophage phiEaSP3 belongs to the Myoviridae family.

[0030] Bacteriophage phiEaSP3 has a head of 93.7±2.48 nm and a long contractile tail of 137.15±4.33 nm.

[0031] Bacteriophage phiEaSP3 has the nucleotide sequence of SEQ ID NO. 1. Bacteriophage phiEaSP3 has a nucleotide sequence of 160.938 bp.

[0032] Bacteriophage phiEaSP3 maintains activity under various environmental conditions. Bacteriophage phiEaSP3 maintains activity over a wide temperature range from 0°C to 50°C and exhibits activity at pH 3 to pH 11. Additionally, bacteriophage phiEaSP3 maintains activity in soil for more than 10 days. Furthermore, bacteriophage phiEaSP3 maintains activity even under conditions of UV-A (λ= 365 nm) exposure. However, activity decreases when exposed to UV-B (λ= 306 nm).

[0033] The present invention provides a composition for inhibiting the proliferation or killing fire blight bacteria comprising bacteriophage phiEaSP3.

[0034] A composition for inhibiting or killing fire blight bacteria containing bacteriophage phiEaSP3 kills only fire blight bacteria due to the host specificity of the bacteriophage and does not affect eukaryotic cells including humans or animals.

[0035] A composition for inhibiting or killing fire blight bacteria containing bacteriophage phiEaSP3 is safe because it does not induce drug resistance or resistance compared to existing antimicrobial substances or antibiotics.

[0036] A composition for inhibiting or killing fire blight bacteria containing bacteriophage phiEaSP3 does not kill the bacteria (Erwinia pyrifoliae) that causes black-shoot blight, which has symptoms similar to fire blight in fruit trees, so it may be used for diagnosing plant diseases.

[0037] The composition for inhibiting or killing fire blight bacteria containing bacteriophage phiEaSP3 according to the present invention can be manufactured in various forms, such as pesticides, disinfectants, and detergents. For example, the composition for inhibiting or killing fire blight bacteria containing bacteriophage phiEaSP3 can be used as a pesticide by spraying it onto fruit trees. For example, the composition for inhibiting or killing fire blight bacteria containing bacteriophage phiEaSP3 can be used as a detergent by treating fruits or containers holding fruits. For example, the composition for inhibiting or killing fire blight bacteria containing bacteriophage phiEaSP3 can be used as a disinfectant by treating agricultural tools, agricultural equipment, agricultural facilities, and the clothing and shoes of farmers and workers.

[0038] The present invention provides a method for controlling fruit trees, comprising the step of treating with a composition for inhibiting the proliferation or killing fire blight pathogens containing bacteriophage phiEaSP3. The method for controlling fruit trees according to the present invention can also be used to prevent fire blight.

[0039] The fruit tree control method of the present invention prevents the emergence of antibiotic-resistant strains and eliminates the problem of antibiotic residue in food, so safe and effective control can be expected.

[0040] The present invention will be explained in more detail below with reference to examples.

[0041] Examples

[0042] Example 1. Isolation of a bacteriophage specific to fire blight pathogens

[0043] Soil and water samples were collected from Chungju, Chungcheongbuk-do, where apple trees were not grown. 20g of soil and water were mixed in a 1:1 ratio and stored at room temperature to obtain an extraction solution. For the analysis, 100μl of culture solution of the fire blight bacterium TS3128 strain was mixed with 5ml of soft agar medium (0.4% LB soft agar) containing a low concentration of agar in LB, and the mixture was poured onto a plate containing 1.5% LB agar medium. 20μl of the extraction solution was dropped onto the solidified soft agar medium and dried. After incubating the plate at 26°C for 12 hours, the result was determined to be positive if a clear plaque was observed. Additionally, soft agar mixed with the same fire blight bacterium as the soft agar medium was prepared. The plaque region was isolated using a sterile tip and dissolved in SM buffer (Sodium-Magnesium). Plaque formation of bacteriophage phiEaSP3 was confirmed using the serial dilution dropping method. The same process was repeated three times to obtain pure bacteriophage phiEaSP3, and the formed plaque is shown in Figure 1.

[0045] Example 2. Confirmation of host-specific susceptibility of bacteriophage phiEaSP3

[0046] The host susceptibility of the isolated bacteriophages was confirmed using the serial dilution drop method. 100 μl of a culture of fire blight bacteria cultured for 12 hours was mixed with 5 ml of 0.4% soft agar medium and poured onto an LB agar plate, then dried at room temperature for 30 minutes. Bacteriophage phiEaSP3 diluted according to the dilution factor was dropped onto the plate, dried at room temperature, and then incubated at 26°C for 12 hours. If lysis spots or single plaques were observed on the plate, it indicated that the bacterial cells had been completely lysed and killed, thus determining that the strain was susceptible to the phage. Table 1 shows the host range of bacteriophage phiEaSP3 susceptible to fire blight bacteria. The bacteriophage phiEaSP3 of the present invention infected all 24 fire blight bacteria used in the test, but failed to infect 8 black twig blight bacteria.

[0047]

[0049] Example 3. Morphological analysis of bacteriophage phiEaSP3

[0050] The morphology of the isolated bacteriophage phiEaSP3 was observed using a transmission electron microscope. High concentrations of bacteriophage (10 6 PFU / 5 µl) was placed on a copper grid for 1 minute, and the solution was removed. Subsequently, negative staining was performed with 2% liquid uranyl acetate for 10 seconds, and the samples were dried. The stained samples were observed using an energy-filtered transmission electron microscope (LIBRA 120, Carl Zeiss) at a voltage of 120 kV. It was confirmed that the observed bacteriophages possessed morphological characteristics, including long, contractile tails (Fig. 2). The head and tail lengths of seven phiEaSP3 phage particles were measured; the head measured 93.7 ± 2.48 nm, and the tail measured 137.15 ± 4.33 nm.

[0052] Example 4. Genome analysis of bacteriophage phiEaSP3

[0053] The bacteriophage proteins were degraded using EDTA, proteinase K, and 10% SDS to expose the DNA. The proteins and DNA were separated by sequential treatment with phenol; phenol:chloroform:isoamyl alcohol 25:24:1; and chloroform, and the DNA was extracted using the ethanol precipitation method. Next Generation Sequencing (NGS) was performed at DNA Link using an Illumina Noveseq 6000 (Illumina, USA) to obtain the complete nucleotide sequence information. Open Reading Frames (ORFs) were analyzed using Genemark.hmm and The RAST server software. The genomic analysis of bacteriophage phiEaSP3 revealed a size of 160,938 bp, and the complete genome sequence is denoted by SEQ ID NO. 1.

[0055] Example 5. Stability test of bacteriophage phiEaSP3 against environmental factors

[0056] The stability of phiEaSP3 was tested against environmental factors (temperature, pH, soil, and UV radiation) that affect the stability of bacteriophages (Fig. 3).

[0057] High concentration of phiEaSP3(10 9 phiEaSP3 (PFU / ml) was stored under three different temperature conditions (37°C, 26°C, and 4°C). Activity was measured on days 0, 1, 3, 5, 7, 14, and 21 when stored at 37°C, and on days 0, 7, 14, and 21 when stored at 26°C and 4°C. The concentration of phiEaSP3 after exposure to each environmental factor was determined using the serial dilution dropwise method. The concentration of phiEaSP3 was maintained at all tested temperatures up to day 7, and a decrease in activity was observed only when stored at 37°C during measurements on days 14 and 21.

[0058] phiEaSP3 lost activity at pH 2 and pH 12, but maintained activity at the remaining pH 3, 4, 5, 7, 10, and 11.

[0059] The activity of phiEaSP3 was measured after mixing soil and a high concentration of phiEaSP3 in a 1.5 ml tube and storing the mixture for 0, 5, 10, and 17 days. The concentration of phiEaSP3 was maintained until 10 days, but after 17 days, 10 6 PFU / ml was expressed. When the activity of phiEaSP3 was measured after exposure to ultraviolet UV-A (λ= 365 nm) and UV-B (λ= 306 nm), the concentration was maintained when exposed to UV-A, but the activity decreased when exposed to UV-B.

[0061] Example 6. Confirmation of the fire blight prevention effect of phiEaSP3 in young apple seedlings

[0062] The preventive effect of bacteriophage phiEaSP3 against fire blight was confirmed in young apple seedlings. Twelve young apple seedlings were divided into four groups (three trees per group). As a control group, sterilized water or a commercially available pesticide (Agrimycin) was applied to the leaves of the seedlings using a brush, and one group was treated with bacteriophage phiEaSP3 (10 8 4 ml of (PFU / ml) was applied to the leaves of 3 cultured seedlings using a brush. After drying at room temperature for 1 hour, fire blight pathogen TS3128 (10 5CFU / ml) was applied to the leaves of three cultured seedlings using a brush. After drying, the apple trees were placed in plastic boxes and stored at room temperature. When fire blight symptoms were observed after 14 days, the three apple cultured seedlings treated only with the fire blight pathogen TS3128 showed symptoms of fire blight, such as branches drying out and breaking and leaves turning brown. On the other hand, no symptoms of fire blight were observed in the three apple trees treated with phiEaSP3, and the condition was identical to that of the control group, confirming that bacteriophage phiEaSP3 can prevent fire blight in apple cultured seedlings (Fig. 4). This implies the potential for using bacteriophage phiEaSP3 as a protective agent against fire blight in fruit tree cultivation environments.

[0064] Depository Name: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Accession Number: KACC87007BP Date of Deposit: 2023-07-13

Claims

Claim 1 Bacteriophage phiEaSP3 (KACC 87007BP) having lytic activity specific to fruit tree fire blight pathogens. Claim 2 In claim 1, a bacteriophage phiEaSP3 having the genome sequence of SEQ ID NO.

1. Claim 3 In claim 1, bacteriophage phiEaSP3 having activity at 0°C to 50°C. Claim 4 In claim 1, bacteriophage phiEaSP3 having activity at pH 3 to 11. Claim 5 A composition for inhibiting the proliferation or killing fire blight bacteria comprising the bacteriophage phiEaSP3 (KACC 87007BP) of any one of claims 1 to 4. Claim 6 A method for controlling fire blight in fruit trees, comprising the step of treating a fruit tree with a composition for inhibiting the proliferation or killing of fire blight bacteria according to claim 5. Claim 7 A method for controlling fire blight pathogens in fruit trees according to claim 6, wherein the fruit tree is any one selected from the group consisting of apple, pear, quince and apricot.

Images