Bacteriophage having bacteriolytic activity against xanthomonas bacteria

Abstract

The present invention addresses the problem of isolating a novel bacteriophage exhibiting bacteriolytic activity specifically against Xanthomonas bacteria and of developing and providing a plant disease control composition having the bacteriophage as an active ingredient, in order to control plant diseases caused by Xanthomonas bacteria.　Provided are: a bacteriolytic agent comprising a bacteriophage that has a novel genomic DNA sequence and exhibits bacteriolytic activity specifically against Xanthomonas bacteria; and a plant disease control composition having the bacteriolytic agent as an active ingredient.

Description

Xanthomonas bacteria lytic bacteriophages

[0001] The present invention relates to a lysant comprising a bacteriophage, a plant disease control composition containing the same, and a method for controlling plant diseases.

[0002] Bacteriophages (often simply abbreviated as "phages" in this specification) are a general term for viruses that infect only bacteria. Many phages, also called lytic phages, specifically adsorb to their target bacteria (hosts), inject their own DNA, and then self-amplify using the bacteria's translation mechanism. Furthermore, they spread the amplified phages by lysing the bacteria, thereby repeatedly infecting new target bacteria (Non-Patent Literature 1).

[0003] Many Xanthomonas bacteria are known to cause plant diseases in various crops and other plants, and copper-based fungicides and antibiotics have been commonly used as conventional control methods. However, due to numerous problems such as efficacy, phytotoxicity, and the potential for disruption of the microbial flora balance, methods using phages have recently attracted attention as a new control method (Non-Patent Literature 2).

[0004] Phages that exhibit lytic activity against Xanthomonas bacteria have been reported, for example, in Patent Documents 1-3 and Non-Patent Document 2. However, because the host range of phages is extremely narrow, and because target bacteria may acquire resistance to specific phages through repeated use of the same type of phage (Non-Patent Document 1), the search for new phages and the discovery of phages with higher lytic activity remain important.

[0005] Japanese Patent Publication No. 2016-32435, Japanese Patent Publication No. 2021-102635, International Publication No. 2017 / 113029

[0006] Sharma S. et al., Folia Microbiol., 2017, 62:17-55Nakayinga R. et al., BMC Microbiology, 2021, 21:291

[0007] To solve the above problems, the inventors focused on bacteriophages. Unlike conventional copper agents and antibiotics, phages, being viruses, are natural products, and therefore no adverse drug reactions have been reported to date. Furthermore, due to their extremely high specificity for hosts, only specific genera or species of bacteria are targeted, and their impact on the balance of the bacterial flora is extremely limited. In addition, they are harmless not only to humans and other animals but also to plants, making them highly safe. Therefore, in order to suppress damage to crops caused by plant diseases resulting from Xanthomonas bacteria, the inventors isolated a novel phage that exhibits broad lytic activity against Xanthomonas bacteria. Furthermore, the inventors aim to provide a composition containing this phage as an active ingredient and to apply it to disease control and detection of pathogenic bacteria.

[0008] The inventors isolated a novel phage from natural wastewater and soil using a method that detects lytic plaques formed on soft agar plates cultured with Xanthomonas bacteria, and analyzed its genome sequence. As a result, it was revealed that the phage possesses a novel genomic DNA sequence with no known related genome sequences and has a distinctive host region.

[0009] The present invention is based on the aforementioned novel findings and provides the following: [1] A bacteriophage comprising a bacteriophage having a genomic DNA sequence consisting of the base sequence shown in any one of SEQ ID NOs: 1, 2, or 6. [2] A bacteriophage comprising a bacteriophage comprising a bacteriophage having a gene in its genomic DNA that encodes a tail fiber protein having recognition activity for target bacteria, consisting of the amino acid sequence shown in SEQ ID NOs: 3. [3] The bacteriophage according to [2], wherein the gene comprises the base sequence shown in SEQ ID NOs: 4. [4] The bacteriophage according to [2] or [3], wherein the base sequence of the genomic DNA comprises the base sequence shown in SEQ ID NOs: 5. [A-1] A bacteriophage comprising a bacteriophage having a genomic DNA sequence containing any of the following (a) to (c): (a) the base sequence shown in SEQ ID NOs: 1 or 2; (b) the base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in SEQ ID NOs: 1 or 2; (c) a base sequence having 96.5% or more sequence identity with the base sequence shown in SEQ ID NOs: 1 or 2. [B-1] A bacteriophage comprising a bacteriophage having in its genomic DNA a gene encoding a tail fiber protein having target bacterial recognition activity, comprising the amino acid sequence shown in any one of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 3; (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 3; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3. [B-2] The bacteriophage according to [B-1], wherein the gene comprises the base sequence shown in any one of (d) to (f) below: (d) the base sequence shown in SEQ ID NO: 4; (e) a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 4; (f) a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 4.[B-3] The lytic agent according to [B-1] or [B-2] wherein the base sequence of the genomic DNA consists of any one of the following base sequences (g) to (k): (g) the base sequence shown in Sequence ID No. 5; (h) the base sequence shown in Sequence ID No. 5 in which one or more bases are added, deleted, and / or substituted in a base sequence other than the gene described in [B-1] or [B-2]; (i) the base sequence shown in Sequence ID No. 5 in which a base sequence other than the gene described in [B-1] or [B-2] has 80% or more sequence identity with the base sequence shown in Sequence ID No. 5; (j) the base sequence shown in Sequence ID No. 5 in which one or more bases are added, deleted, and / or substituted; (k) the base sequence shown in Sequence ID No. 5 has 90% or more sequence identity. [C-1] A lytic agent comprising a bacteriophage having a genomic DNA sequence containing any of the following (a) to (c): (a) the sequence shown in Sequence ID No. 6, (b) a sequence in which one or more bases are added, deleted, and / or substituted in the sequence shown in Sequence ID No. 6, and (c) a sequence having 90% or more sequence identity with the sequence shown in Sequence ID No. 6. [5] A lytic agent according to any of [1] to [4] that exhibits lytic activity against bacteria of the genus Xanthomonas. [A-5] A lytic agent according to [A-1] that exhibits lytic activity against bacteria of the genus Xanthomonas. [A-6] The lytic agent according to [A-5], wherein the Xanthomonas bacteria is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas citri, Xanthomonas campestris, and Xanthomonas cucurbitae. [B-5] The lytic agent according to any one of [B-1] to [B-3], which exhibits lytic activity against Xanthomonas bacteria.[B-6] The lytic agent according to [B-5], wherein the Xanthomonas bacteria is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, and Xanthomonas citri. [C-5] The lytic agent according to [C-1], exhibiting lytic activity against Xanthomonas bacteria. [C-6] The lytic agent according to [C-5], wherein the Xanthomonas bacteria is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, and Xanthomonas citri. [6] A composition comprising the lytic agent according to any one of [1] to [C-6] as an active ingredient. [7] A plant disease control composition comprising the composition described in [6]. [8] The plant disease control composition according to [7], wherein the plant disease is a plant disease caused by a bacteriophage of the genus Xanthomonas. [9] The plant disease control composition according to [7] or [8], comprising another bacteriophage that exhibits lytic activity against a bacteriophage of the genus Xanthomonas.

[10] A method for controlling a plant disease, comprising a contact step of contacting a target plant with the plant disease control composition described in any of [7] to [9].

[11] A method for identifying a bacteriophage of the genus Xanthomonas, comprising a culture step of culturing a test bacterium isolated from plant tissue affected by a plant disease to obtain a culture, a mixing step of mixing the culture with the lytic agent described in [5] to obtain a mixture, a mixture culture step of culturing the mixture under predetermined conditions, and a determination step of determining that the test bacterium is a bacteriophage of the genus Xanthomonas when the test bacterium is lytic after the mixture culture step.

[12] The method according to

[11] , wherein in the mixture culture step, the mixture further comprises a soft agar-containing liquid medium, and the mixture is cultured on a solid medium.

[13] The method according to

[11] , wherein in the culture step, the culture comprises a soft agar-containing liquid medium, and the culture is cultured on a solid medium.

[14] The method according to any one of

[11] to

[13] , further comprising an isolation step of isolating the test bacteria from plant tissue infected with a plant disease before the culture step.This specification encompasses the disclosures of Japanese Patent Application Nos. 2025-056387, 2025-056385, 2025-056384, and 2025-056386, which form the basis of the priority claim of this application.

[0010] The bactericidal agent of the present invention and the composition containing it as an active ingredient can lyse specific target bacteria.

[0011] The plant disease control composition of the present invention can prevent and suppress diseases caused by specific target bacteria.

[0012] This figure shows the lytic activity of the first bacteriophage obtained in Example 1. A is a figure obtained by culturing Xanthomonas bacteria and Pseudomonas fluorescens (a control) on an agar plate, then dropping the first phage purified solution into the center of the plate and allowing it to be cultured statically. B is a plate figure corresponding to A, showing the bacteria shown in Table 2 spread on each plate. In the figure, "a-1" to "a-4" show the results when a phage with the genomic DNA sequence of SEQ ID NO: 2 is used as the first bacteriophage, and "b-1" to "b-4" show the results when a phage with the genomic DNA sequence of SEQ ID NO: 1 is used as the first bacteriophage. This figure shows the lytic activity of the second bacteriophage obtained in Example 2. A is a figure obtained by culturing Xanthomonas bacteria and Pseudomonas fluorescens (a control) spread on an agar plate, then dropping the second phage purified solution into the center of the plate and allowing it to be cultured statically. Figure B is a plate diagram corresponding to A, showing the bacteria shown in Table 2 spread on each plate. This figure shows the lytic activity of the third bacteriophage obtained in Example 3. Figure A shows the results after culturing Xanthomonas bacteria and the control Pseudomonas fluorescens on an agar plate, then dropping the third phage purified solution onto the center of the plate and allowing it to be cultured statically. Figure B is a plate diagram corresponding to A, showing the bacteria shown in Table 2 spread on each plate. This figure shows the lytic activity of the bacteriophage against the Xanthomonas bacterium Xanthomonas campestris pv. vesicatoria (MAFF No. 301256). Figure A shows the results after culturing Xanthomonas bacteria shown in Table 4 spread on an agar plate, then dropping the phage purified solution onto the plate and allowing it to be cultured statically. Figure B is a plate diagram corresponding to A, showing the phages dropped at various positions on the plate.In the figure, "a" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 10 in WO 2023 / 191074 was dropped, "b" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 was dropped, "c" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 14 in WO 2023 / 191074 was dropped, "d" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 17 in WO 2023 / 191074 was dropped, "e" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 19 in WO 2023 / 191074 was dropped, "f" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 23 in WO 2023 / 191074 was dropped, "g" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 29 in WO 2023 / 191074 was dropped, and "h" is WO "i" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 35 in WO 2023 / 191074 was dropped, "j" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 41 in WO 2023 / 191074 was dropped, "k" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 was dropped, "l" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 was dropped, "m" is WO The diagram shows the positions where the phage with the genomic DNA sequence of Sequence ID No. 3 in 2024 / 248029 was dropped, "n" indicates the position where the first phage obtained in Example 1 (having the genomic DNA sequence of Sequence ID No. 2) was dropped, "o" indicates the position where the second phage obtained in Example 2 was dropped, and "p" indicates the position where the third phage obtained in Example 3 was dropped. The diagram shows the lytic activity of bacteriophages against the bacterium Xanthomonas arboricola pv. juglandis (MAFF No. 212147). A is a diagram of the Xanthomonas bacteria shown in Table 4, which were cultured on an agar plate, after which the phage purified solution was dropped onto the plate and allowed to culture statically. B is a diagram of the plate corresponding to A, showing the phages dropped at each position on the plate.In the figure, "a" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 10 in WO 2023 / 191074 was dropped, "b" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 was dropped, "c" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 14 in WO 2023 / 191074 was dropped, "d" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 17 in WO 2023 / 191074 was dropped, "e" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 19 in WO 2023 / 191074 was dropped, "f" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 23 in WO 2023 / 191074 was dropped, "g" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 29 in WO 2023 / 191074 was dropped, and "h" is WO "i" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 35 in WO 2023 / 191074 was dropped, "j" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 41 in WO 2023 / 191074 was dropped, "k" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 was dropped, "l" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 was dropped, "m" is WO The diagram shows the positions where the phage with the genomic DNA sequence of Sequence ID No. 3 in 2024 / 248029 was dropped, "n" indicates the position where the first phage obtained in Example 1 (having the genomic DNA sequence of Sequence ID No. 2) was dropped, "o" indicates the position where the second phage obtained in Example 2 was dropped, and "p" indicates the position where the third phage obtained in Example 3 was dropped. This diagram shows the lytic activity of bacteriophages against the bacterium Xanthomonas citri subsp. citri (MAFF No. 302101). A is a diagram of the Xanthomonas bacteria shown in Table 4, which were cultured on an agar plate, after which the phage purified solution was dropped onto the plate and allowed to culture statically. B is a plate diagram corresponding to A, showing the phages dropped at each position on the plate.In the figure, "a" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 10 in WO 2023 / 191074 was dropped, "b" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 was dropped, "c" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 14 in WO 2023 / 191074 was dropped, "d" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 17 in WO 2023 / 191074 was dropped, "e" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 19 in WO 2023 / 191074 was dropped, "f" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 23 in WO 2023 / 191074 was dropped, "g" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 29 in WO 2023 / 191074 was dropped, and "h" is WO "i" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 35 in WO 2023 / 191074 was dropped, "j" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 41 in WO 2023 / 191074 was dropped, "k" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 was dropped, "l" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 was dropped, "m" is WO The diagram shows the positions where the phage with the genomic DNA sequence of Sequence ID No. 3 (2024 / 248029) was dropped, "n" indicates the position where the first phage obtained in Example 1 (having the genomic DNA sequence of Sequence ID No. 2) was dropped, "o" indicates the position where the second phage obtained in Example 2 was dropped, and "p" indicates the position where the third phage obtained in Example 3 was dropped. This diagram shows the lytic activity of bacteriophages against the bacterium Xanthomonas citri subsp. citri (MAFF No. 311131). A is a diagram of the Xanthomonas bacteria shown in Table 4, which were cultured on an agar plate, after which the phage purified solution was dropped onto the plate and allowed to culture statically. B is a diagram of the plate corresponding to A, showing the phages dropped at each position on the plate.In the figure, "a" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 10 in WO 2023 / 191074 was dropped, "b" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 was dropped, "c" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 14 in WO 2023 / 191074 was dropped, "d" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 17 in WO 2023 / 191074 was dropped, "e" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 19 in WO 2023 / 191074 was dropped, "f" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 23 in WO 2023 / 191074 was dropped, "g" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 29 in WO 2023 / 191074 was dropped, and "h" is WO "i" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 35 in WO 2023 / 191074 was dropped, "j" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 41 in WO 2023 / 191074 was dropped, "k" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 was dropped, "l" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 was dropped, "m" is WO The diagram shows the positions where the phage with the genomic DNA sequence of Sequence ID No. 3 in 2024 / 248029 was dropped, "n" indicates the position where the first phage obtained in Example 1 (having the genomic DNA sequence of Sequence ID No. 2) was dropped, "o" indicates the position where the second phage obtained in Example 2 was dropped, and "p" indicates the position where the third phage obtained in Example 3 was dropped. This diagram shows the lytic activity of bacteriophages against the bacterium Xanthomonas citri subsp. citri (MAFF No. 673001). A is a diagram of the Xanthomonas bacteria shown in Table 4, which were cultured on an agar plate, after which the phage purified solution was dropped onto the plate and allowed to culture statically. B is a plate diagram corresponding to A, showing the phages dropped at each position on the plate.In the figure, "a" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 10 in WO 2023 / 191074 was dropped, "b" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 was dropped, "c" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 14 in WO 2023 / 191074 was dropped, "d" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 17 in WO 2023 / 191074 was dropped, "e" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 19 in WO 2023 / 191074 was dropped, "f" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 23 in WO 2023 / 191074 was dropped, "g" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 29 in WO 2023 / 191074 was dropped, and "h" is WO "i" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 35 in WO 2023 / 191074 was dropped, "j" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 41 in WO 2023 / 191074 was dropped, "k" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 was dropped, "l" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 was dropped, "m" is WO The diagram shows the positions where the phage with the genomic DNA sequence of Sequence ID No. 3 in 2024 / 248029 was dropped, "n" indicates the position where the first phage obtained in Example 1 (having the genomic DNA sequence of Sequence ID No. 2) was dropped, "o" indicates the position where the second phage obtained in Example 2 was dropped, and "p" indicates the position where the third phage obtained in Example 3 was dropped. This diagram shows the lytic activity of bacteriophages against the bacterium Xanthomonas citri subsp. citri (MAFF No. 673008). A is a diagram of the Xanthomonas bacteria shown in Table 4, which were cultured on an agar plate, after which the phage purified solution was dropped onto the plate and allowed to culture statically. B is a plate diagram corresponding to A, showing the phages dropped at each position on the plate.In the figure, "a" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 10 in WO 2023 / 191074 was dropped, "b" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 was dropped, "c" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 14 in WO 2023 / 191074 was dropped, "d" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 17 in WO 2023 / 191074 was dropped, "e" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 19 in WO 2023 / 191074 was dropped, "f" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 23 in WO 2023 / 191074 was dropped, "g" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 29 in WO 2023 / 191074 was dropped, and "h" is WO "i" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 35 in WO 2023 / 191074 was dropped, "j" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 41 in WO 2023 / 191074 was dropped, "k" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 was dropped, "l" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 was dropped, "m" is WO The diagram shows the positions where the phage with the genomic DNA sequence of Sequence ID No. 3 in 2024 / 248029 was dropped, "n" indicates the position where the first phage obtained in Example 1 (having the genomic DNA sequence of Sequence ID No. 2) was dropped, "o" indicates the position where the second phage obtained in Example 2 was dropped, and "p" indicates the position where the third phage obtained in Example 3 was dropped. This diagram shows the lytic activity of bacteriophages against the bacterium Xanthomonas citri subsp. citri (MAFF No. 673013). A is a diagram of the Xanthomonas bacteria shown in Table 4, which were cultured on an agar plate, after which the phage purified solution was dropped onto the plate and allowed to culture statically. B is a diagram of the plate corresponding to A, showing the phages dropped at each position on the plate.In the figure, "a" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 10 in WO 2023 / 191074 was dropped, "b" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 was dropped, "c" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 14 in WO 2023 / 191074 was dropped, "d" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 17 in WO 2023 / 191074 was dropped, "e" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 19 in WO 2023 / 191074 was dropped, "f" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 23 in WO 2023 / 191074 was dropped, "g" is the location where a phage with the genomic DNA sequence of SEQ ID NO: 29 in WO 2023 / 191074 was dropped, and "h" is WO "i" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 35 in WO 2023 / 191074 was dropped, "j" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 41 in WO 2023 / 191074 was dropped, "k" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 was dropped, "l" is the location where the phage with the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 was dropped, "m" is WO The diagram shows the positions where the phage with the genomic DNA sequence of Sequence ID No. 3 in 2024 / 248029 was dropped, "n" indicates the position where the first phage obtained in Example 1 (having the genomic DNA sequence of Sequence ID No. 2) was dropped, "o" indicates the position where the second phage obtained in Example 2 was dropped, and "p" indicates the position where the third phage obtained in Example 3 was dropped. This diagram shows the lytic activity of the first bacteriophage obtained in Example 1 against Xanthomonas citri subsp. citri. A is a diagram of Xanthomonas bacteria shown in Table 5 cultured on an agar plate, after which the first phage purified solution was dropped into the center of the plate and allowed to culture statically. B is a plate diagram corresponding to A, showing the phages dropped at each position on the plate.In the figure, "1" shows a plate on which bacteria of MAFF No. 673034 were spread, "2" shows a plate on which bacteria of MAFF No. 302101 were spread, "3" shows a plate on which bacteria of MAFF No. 673005 were spread, "4" shows a plate on which bacteria of MAFF No. 311134 were spread, "5" shows a plate on which bacteria of MAFF No. 311131 were spread, and "6" shows a plate on which bacteria of MAFF No. 673034 were spread. In the figure, "a" shows the position where the phage having the genomic DNA sequence of SEQ ID NO: 1 was dropped, and "b" shows the position where the phage having the genomic DNA sequence of SEQ ID NO: 2 was dropped. This figure shows the lytic activity of the first bacteriophage (having the genomic DNA sequence of SEQ ID NO: 2) obtained in Example 1 against other Xanthomonas bacteria. A is a figure on which Xanthomonas bacteria were cultured on an agar plate, and then the first phage purified solution was dropped into the center of the plate and allowed to stand. Figure B is a plate diagram corresponding to A, showing the bacteria shown in Table 6 spread on each plate. This figure shows the lytic activity of the second bacteriophage obtained in Example 2 against Xanthomonas citri subsp. citri and other Xanthomonas bacteria. Figure A shows the result of culturing Xanthomonas bacteria on an agar plate, then dropping the second phage purified solution into the center of the plate and allowing it to be cultured statically. Figure B is a plate diagram corresponding to A, showing the bacteria shown in Table 7 spread on each plate. This figure shows the lytic activity of the second bacteriophage obtained in Example 2 against other Xanthomonas bacteria. Figure A shows the result of culturing Xanthomonas bacteria on an agar plate, then dropping the second phage purified solution into the center of the plate and allowing it to be cultured statically. Figure B is a plate diagram corresponding to A, showing the bacteria shown in Table 8 spread on each plate. This figure shows the lytic activity of the third bacteriophage obtained in Example 3 against Xanthomonas citri subsp. citri and other Xanthomonas bacteria. Figure A shows the results after culturing Xanthomonas bacteria on an agar plate, dropping the purified third phage solution into the center of the plate, and allowing it to be cultured statically. Figure B is the plate diagram corresponding to A, showing the bacteria shown in Table 9 spread on each plate.This figure shows the relative residual titers of the cryopreserved groups of bacteriophages 1-3. The relative residual titer is the ratio to the titer of the 4°C storage group. In the figure, the dashed line represents a relative residual titer of 100%, indicating a level where there is no change in titer compared to the 4°C storage group. Error bars indicate the standard deviation. This graph shows the results of the disease control efficacy test for tomato bacterial spot disease conducted in Example 9. The average disease incidence rate is shown as a relative value to the untreated group. In the figure, "a" represents the first phage obtained in Example 1 (having the genomic DNA sequence of SEQ ID NO: 2), "b" represents the second phage obtained in Example 2 (having the genomic DNA sequence of SEQ ID NO: 5), "c" represents the phage having the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 (SEQ ID NO: 7), "d" represents the phage having the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 (SEQ ID NO: 8), and "e" represents the phage having the genomic DNA sequence of SEQ ID NO: 1 in WO 2024 / 248029 (SEQ ID NO: 9). In the figure, " / " indicates that the preceding and succeeding phages are used in combination. For example, "a / b" indicates that the first and second phages are used in combination. This graph shows the results of the disease control efficacy test for broccoli black rot conducted in Example 10. The average disease incidence rate is shown as a relative value to the untreated group. In the figure, "a" represents the first phage obtained in Example 1 (having the genomic DNA sequence of SEQ ID NO: 2), "b" represents the second phage obtained in Example 2 (having the genomic DNA sequence of SEQ ID NO: 5), "c" represents the third phage obtained in Example 3 (having the genomic DNA sequence of SEQ ID NO: 6), "d" represents the phage having the genomic DNA sequence of SEQ ID NO: 47 (SEQ ID NO: 7) in WO 2023 / 191074, "e" represents the phage having the genomic DNA sequence of SEQ ID NO: 44 (SEQ ID NO: 8) in WO 2023 / 191074, "f" represents the phage having the genomic DNA sequence of SEQ ID NO: 2 (SEQ ID NO: 10) in WO 2024 / 248029, and "g" represents the phage having the genomic DNA sequence of SEQ ID NO: 3 (SEQ ID NO: 11) in WO 2024 / 248029. In the figure, " / " indicates that the preceding and succeeding phages are used in combination.For example, "a / b / c" indicates that the first phage, the second phage, and the third phage are being used in combination.

[0013] 1. Lysolytic Agent 1-1. Overview The first aspect of the present invention is a lysolytic agent. The lysolytic agent of the present invention comprises a bacteriophage having a genome sequence containing a specific base sequence. The lysolytic agent of the present invention exhibits specific lytic activity against target bacteria that may be pathogenic bacteria of plant diseases.

[0014] 1-2. Definitions The terms used herein are defined below.

[0015] In this specification, "lytic agent" means a drug comprising a bacteriophage that has lytic activity against target bacteria.

[0016] Bacteria, along with archaea and eukaryotes, are one of the three major groups of organisms that make up the entire biological world. Bacteria consist of cells without a nucleus and can self-replicate if nutrients are available. Bacterial names are indicated by the family, genus, and species levels, according to the International Code of Bacterial Nomenclature.

[0017] In this specification, "target bacteria" refers to host bacteria that can be targeted by phages constituting the lytic agent of the present invention, or phages contained in the composition and plant disease control composition of the present invention. For example, bacteria having membrane surface receptors on the extracellular membrane that are recognized by the above-mentioned phages. "Membrane surface receptors" are sites where, for example, the tail and tail fibers of phages bind, and are composed of proteins, lipopolysaccharides, or fimbriae present in the outer layer of the bacterial outer membrane. A specific example of target bacteria in this specification is bacteria of the genus Xanthomonas.

[0018] "Xanthomonas bacteria" are bacteria belonging to the genus Xanthomonas. Xanthomonas bacteria generally produce a yellow pigment called xanthomonadin, and many are known as plant pathogenic bacteria. Below the species level, there are also subtypes and pathovars, which are indicated by "subsp." or "pv." after the bacterial name, respectively. The smallest unit of classification is the strain, which refers to a group of cells considered to be genetically uniform. Table 1 below shows representative Xanthomonas bacteria, their host plants, and the plant diseases they cause.

[0019]

[0020] While not limited to these, among bacteria of the genus Xanthomonas, Xanthomonas arboricola, Xanthomonas citri, Xanthomonas campestris, and Xanthomonas cucurbitae are particularly preferred as target bacteria of the present invention. Specific examples of Xanthomonas arboricola include Xanthomonas arboricola pv. pruni, whose prototype is pruni, and Xanthomonas arboricola pv. juglandis, whose prototype is juglandis. A specific example of Xanthomonas citri is Xanthomonas citri subsp. citri. Specific examples of Xanthomonas campestris include Xanthomonas campestris pv. vesicatoria, whose prototype is vesicatoria; Xanthomonas campestris pv. raphani, whose prototype is raphani; and Xanthomonas campestris pv. campestris, whose prototype is campestris.

[0021] A "bacteriophage" (as mentioned above, often simply abbreviated as "phage" in this specification) is a general term for viruses that infect bacteria. A typical phage consists of three parts: the head, the tail, and the tail fiber. The head is composed of a capsomere, an outer covering protein, and consists of an icosahedral capsid (viral shell) that contains the phage's genomic DNA within its internal space. The tail has a tubular structure consisting of a tail tube protein and a sheath protein that covers it. One end of the tail is connected to the head, and the other end is connected to the tail fiber. The tail functions as an introduction tube that injects the head's genomic DNA into the host bacterial cell. The tail and tail fiber are composed of several fibrous structures made of tail fiber protein. The tail and tail fiber are responsible for host recognition and adsorption functions, recognizing receptors present on the outer membrane surface of the host bacterium and adsorbing to its cell surface. Phages exhibit extremely high host specificity, and their characteristics are based on the function of their tails and tail fibers. Since phages do not infect eukaryotes, drugs using phages are harmless to humans, animals, and plants. Phages are broadly classified into "lytic cycle," "lysogenic cycle," and "lytic / lysogenic cycle" based on their mode of infection. The lysogenic cycle involves integrating its own DNA into the chromosome of the target bacterium without lysing it, and multiplying along with the bacterial growth. On the other hand, the lytic cycle involves self-replicating within the host bacterial cell, then lysing the host bacterium and releasing a large number of progeny phages. The phages of this invention are virulent phages that undergo the lytic cycle.

[0022] As mentioned above, "tail fiber proteins" are proteins that make up the tail fibers of phages. Tail fiber proteins are known to play an important role in the specificity of host recognition and adsorption capacity of the tail and tail fibers (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16:760-773).

[0023] A "tail fiber gene" refers to a gene contained in the genomic DNA of a phage that codes for the aforementioned tail fiber protein.

[0024] "Lyolysis" refers to the phenomenon of destroying the cell membrane of bacteria. As mentioned above, this phenomenon is mainly observed in the infection mode of virulent phages. Bacteria die through lysis. Lysis begins when a phage specifically adsorbs to a target bacterium and injects its own DNA into the target bacterium's cell via its tail. Subsequently, it uses the bacterium's translation mechanism to replicate itself and produce a large number of progeny phages, which then lyse the bacterium and release the progeny phages into the environment.

[0025] In this specification, "plant disease" refers to a general term for diseases that occur in plants. Plant diseases include those caused by infectious pathogens such as viruses, bacteria, filamentous fungi, actinomycetes, viroids, phytoplasmas, nematodes, mites, or insects, as well as those caused by non-infectious pathogens such as lack or excess of nutrients or water, or phytotoxicity. In this specification, unless otherwise specified, plant diseases refer to diseases caused by bacteria, i.e., plant pathogenic bacteria. In this specification, unless otherwise specified, plant pathogenic bacteria refer to the aforementioned target bacteria, such as bacteria of the genus Xanthomonas.

[0026] In this specification, "control" means prevention or treatment (extermination) (from the Japan Agricultural Chemicals Manufacturers Association website). Therefore, in this specification, "plant disease control" means prevention of plant diseases, especially target bacteria, or treatment of plant diseases caused by target bacteria.

[0027] In this specification, "target plant" refers to a plant to which the plant disease control composition of the present invention, described later, is applied. This plant includes plants that have developed a specific plant disease due to infection by the target bacteria, or plants that are at risk of infection by the target bacteria.

[0028] 1-3. Composition The lytic agent of the present invention consists of a bacteriophage. <First phage> The first phage is characterized by having a genomic DNA sequence containing a specific base sequence and exhibits specific lytic activity against target bacteria.

[0029] (1-1) Genomic DNA The first phage has a genome DNA sequence which is a 63,913 bp sequence shown in SEQ ID NO: 1, a 63,780 bp sequence shown in SEQ ID NO: 2, or a sequence in which one or more bases are added, deleted, and / or substituted in the sequence shown in SEQ ID NO: 1 or 2, or a sequence which has 96.5%, 97%, 98%, 99%, 99.5% or more sequence identity with the sequence shown in SEQ ID NO: 1 or 2, or a sequence which has 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity when aligned with the sequence shown in SEQ ID NO: 1 or 2, or a sequence which further has a sequence which hybridizes under highly stringent conditions to a sequence complementary to the sequence shown in SEQ ID NO: 1 or 2.

[0030] In long genomic DNA sequences, it is not easy to align the entire range to use as a comparison range. It is most preferable that the comparison range (sometimes referred to as Query Cover) covers 100% of the entire genome of the first phage, but it may also be 99% or more, 98.5% or more, 98.2% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, or 90% or more.

[0031] In this specification, "multiple items" means 2 to 10 items, for example, 2 to 7 items, 2 to 5 items, 2 to 4 items, or 2 to 3 items.

[0032] In this specification, "(base) sequence identity" refers to a numerical value indicating the proportion of regions within the comparison range of two base sequences where the types of bases are identical. Even if the lengths of the two base sequences are different, base sequence identity can be calculated by aligning them to maximize the degree of base agreement within the comparison range. While not limited to this, BLAST is a representative algorithm for performing such analysis. BLAST is available in various software and web services. For example, base sequence identity can be easily calculated using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ), the NCBI-provided BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), etc. In addition to BLAST, there are other algorithms such as FASTA, which can be used if a reasonable identity can be calculated, and the method of use is not particularly limited.

[0033] The aforementioned "highly stringent conditions" refer to environmental conditions that make nonspecific hybridization unlikely. Under highly stringent conditions, nucleic acids with the target base sequence can form hybrids, but nucleic acids with nonspecific base sequences cannot substantially form hybrids. Generally, highly stringent conditions refer to conditions with low salt concentration and high temperature. Low salt concentration refers to, for example, 15 mM to 750 mM, preferably 15 mM to 500 mM, 15 mM to 300 mM, or 15 mM to 200 mM. High temperature refers to, for example, 50 to 68°C, or 55 to 70°C. A specific example of highly stringent conditions is washing after hybridization at 65°C with 0.1 × SSC and 0.1% SDS.

[0034] From another perspective, since phages are viruses, it is conceivable that mutations such as substitutions, deletions, and insertions may occur in the genomic DNA when amplifying the phages of the present invention. As long as the extent of these mutations is within the range described above for the entire genomic DNA and the lytic function of the phage is maintained, the phages are included in the present invention even if mutations occur.

[0035] Depending on the software or analysis server, sequence identity may be indicated by metrics such as Average Nucleotide Identity (ANI), and these may also be used. However, with long phage genome DNA, it is not easy to align and compare the entire range. Therefore, the sequence identity may be present only within the range automatically aligned by the aforementioned software or web service. For example, in analysis using the NCBI-provided BLAST server, the query sequence and subject sequence are automatically aligned (often referred to as "aligned arrangement" in this specification) within the largest possible range, and the comparison range is determined. Sequence identity within this comparison range is calculated, and the ratio of the comparison range to the entire range of the query sequence may be calculated as a value called Query Cover.

[0036] These values ​​may change depending on how the query array and target array are chosen. This point will be briefly explained using the example of a first array composed of array A and a second array composed of array A and array B of the same length. If the shorter first array is used as the query array and the longer second array as the target array, the comparison range is array A, and since array A is the total length of the first array, the calculated Query Cover value will be 100%. Also, since array A in the first array and array A in the second array are identical, the array identity value will be 100%. On the other hand, if the longer second array is used as the query array and the shorter first array as the target array, the array identity value will be 100% as before, but since array A occupies only half of the second array, the Query Cover value will be 50%.

[0037] In such cases, the sequence identity of the base sequence over the entire range of the aligned base sequence (overall sequence identity) can be estimated based on the results. For example, the estimated value of the overall sequence identity can be obtained by multiplying the Query Cover value by the value of sequence identity within the aligned range (sequence identity within the aligned range). In this case, further modifications may be made to improve the accuracy of the estimate, such as including the expected sequence identity in ranges other than the aligned range.

[0038] Incidentally, when phage genomic DNA is packaged, it may be either linear or circular. Furthermore, in next-generation genome sequencer analysis, after fragmenting genomic DNA, the base sequence of each individual fragment is read, and the sequence is determined through analysis that aligns these reads. In the case of phages, de novo assembly, in which sequences are assembled without using a reference genomic DNA sequence, is often performed. Therefore, it is difficult to uniquely determine the start and end points of the analyzed genome (Merrill, B.D., et al. BMC Genomics, 2016 17, 679). The start and end points of the compared genomic sequences do not need to be matched, and this is automatically taken into account in analysis using software or analysis servers.

[0039] Although not particularly limited, among the Xanthomonas bacteria described above in the definition section, Xanthomonas arboricola, Xanthomonas citri, Xanthomonas campestris, and Xanthomonas cucurbitae are preferred as target bacteria for the first phage. Specific examples of Xanthomonas arboricola suitable as target bacteria for the first phage include Xanthomonas arboricola pv. pruni, whose pathovar is pruni, and Xanthomonas arboricola pv. juglandis, whose pathovar is juglandis. As a specific example of Xanthomonas citri suitable as a target bacterium for the first phage, Xanthomonas citri subsp. citri may be mentioned. Specific examples of Xanthomonas campestris suitable as a target bacterium for the first phage include Xanthomonas campestris pv. vesicatoria, whose pathovar is vesicatoria, and Xanthomonas campestris pv. campestris, whose pathovar is campestris.

[0040] (1-2) Effect The first phage can exhibit lytic activity against a wide range of species of Xanthomonas bacteria, particularly species such as Xanthomonas citri, and can be applied to plant disease control.

[0041] <Second phage> Said second phage is characterized in that it comprises, in its genomic DNA, a gene encoding a tail fiber protein consisting of a specific amino acid sequence, and exhibits specific lytic activity against a target bacterium.

[0042] (2-1) Tail Fiber Protein The tail fiber protein consists of the amino acid sequence represented by SEQ ID NO: 3 composed of 1631 amino acid residues, an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3. Any tail fiber protein is characterized by having target bacterium-specific recognition activity.

[0043] The term "(amino acid) substitution" refers to substitution within a group of conserved amino acids having similar properties such as charge, side chain, polarity, aromaticity, etc., among the 20 types of amino acids that constitute natural proteins. Examples thereof include substitutions within the group of uncharged polar amino acids having low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), the group of branched-chain amino acids (Leu, Val, Ile), the group of neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), the group of neutral amino acids having hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), the group of acidic amino acids (Asp, Glu), the group of basic amino acids (Arg, Lys, His), and the group of aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because it is known that they are less likely to cause changes in the properties of the polypeptide.

[0044] Furthermore, in this specification, "amino acid sequence identity" refers to a numerical value indicating the proportion of sites within the comparison range of two amino acid sequences where the types of amino acid residues are identical. Even if the lengths of the two amino acid sequences are different, amino acid sequence identity can be calculated by aligning them so that the degree of amino acid agreement within the comparison range is maximized. While not limited to this, algorithms such as BLAST, as previously described, can be used to perform such analysis.

[0045] (2-2) Tail fiber gene The second bacteriophage contains a tail fiber gene in its genomic DNA, which consists of a base sequence encoding the tail fiber protein.

[0046] Specific examples of tailfiber gene sequences include, for example, the sequence shown in Sequence ID No. 4, or a sequence in which one or more bases are added, deleted, and / or substituted in the sequence shown in Sequence ID No. 4, a sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the sequence shown in Sequence ID No. 4, or a sequence that hybridizes under highly stringent conditions to a sequence complementary to the sequence shown in Sequence ID No. 4. Note that the sequence of Sequence ID No. 4 is a partial sequence of the complementary strand of genomic DNA having the sequence of Sequence ID No. 5.

[0047] The proteins encoded by the aforementioned tail fiber genes all possess lytic activity against target bacteria.

[0048] (2-3) Genomic DNA The DNA sequence of the phage's genome is a 170,031 bp nucleotide sequence shown in Sequence ID No. 5, a nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence in a region other than the tail fiber gene in the nucleotide sequence shown in Sequence ID No. 5, and a nucleotide sequence in which the nucleotide sequence in a region other than the tail fiber gene in the nucleotide sequence shown in Sequence ID No. 5 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 5, and one or This includes a base sequence in which multiple bases are added, deleted, and / or substituted, or a base sequence having 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more sequence identity with the base sequence shown in Sequence ID No. 5, or a base sequence having 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more sequence identity when aligned with the base sequence shown in Sequence ID No. 5, or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in Sequence ID No. 5.

[0049] In long genomic DNA sequences, it is not easy to use the entire range as the comparison range. It is most preferable that the range to be compared (hereinafter sometimes referred to as Query Cover) covers 100% of the total genome length of the second phage, but it may also be 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, or 90% or more.

[0050] While not particularly limited, among the Xanthomonas species mentioned above in the definition section, Xanthomonas arboricola, Xanthomonas citri, and Xanthomonas campestris are preferred as target bacteria for the second phage. Specific examples of Xanthomonas arboricola suitable as target bacteria for the second phage include Xanthomonas arboricola pv. pruni, whose prototype is pruni, and Xanthomonas arboricola pv. juglandis, whose prototype is juglandis. A specific example of Xanthomonas citri suitable as a target bacteria for the second phage is Xanthomonas citri subsp. citri. Specific examples of Xanthomonas campestris suitable as target bacteria for the second phage include Xanthomonas campestris pv. vesicatoria, whose prototype is vesicatoria; Xanthomonas campestris pv. raphani, whose prototype is raphani; and Xanthomonas campestris pv. campestris, whose prototype is campestris.

[0051] (2-4) The second phage can exhibit lytic activity against a wide range of Xanthomonas species, particularly Xanthomonas citri, and can be applied to plant diseases.

[0052] <Third Phage> This third phage is characterized by having a genomic DNA sequence containing a specific base sequence, and exhibits specific lytic activity against target bacteria.

[0053] (3-1) Genomic DNA The DNA sequence of the third phage's genome is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 94.5% or more, 94.7% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more of the 45,162 bp base sequence shown in SEQ ID NO: 6, or a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 6, or 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 94.5% or more, 94.7% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more The present invention relates to a nucleotide sequence having sequence identity, a nucleotide sequence having sequence identity of 94.7% or more, 95% or more, 96% or more, 96.5% or more, 97.0% or more, 98.0% or more, 98.5% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more when aligned with the nucleotide sequence shown in Sequence ID No. 6, and a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 6.

[0054] In long genomic DNA sequences, it is not easy to use the entire range as the comparison range. It is most preferable that the range to be compared (hereinafter sometimes referred to as Query Cover) covers 100% of the total genome length of the third phage, but it may also be 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, 90% or more, 89% or more, or 88% or more.

[0055] While not particularly limited, among the Xanthomonas species mentioned above in the definition section, Xanthomonas arboricola, Xanthomonas citri, and Xanthomonas campestris are preferred as target bacteria for the third phage. Specific examples of Xanthomonas arboricola suitable as target bacteria for the third phage include Xanthomonas arboricola pv. pruni, whose prototype is pruni, and Xanthomonas arboricola pv. juglandis, whose prototype is juglandis. A specific example of Xanthomonas citri suitable as a target bacteria for the third phage is Xanthomonas citri subsp. citri. Specific examples of Xanthomonas campestris suitable as target bacteria for the third phage include Xanthomonas campestris pv. vesicatoria, whose prototype is vesicatoria, and Xanthomonas campestris pv. campestris, whose prototype is campestris.

[0056] (3-2) The third phage exhibits lytic activity against a wide range of Xanthomonas species, particularly Xanthomonas citri, and can be applied to plant diseases.

[0057] 2. Composition for controlling plant diseases 2-1. Overview A second aspect of the present invention is a composition, particularly a composition usable for controlling plant diseases. The composition of the present invention is characterized by containing the lysant described in the first aspect as an active ingredient.

[0058] According to the composition of the present invention, when used for controlling plant diseases, it is possible to provide a sustainable pesticide for bacterial plant diseases that is safe for humans, does not cause phytotoxicity to the environment, and can specifically prevent or treat the target plant disease.

[0059] In this specification, the term "plant disease control composition" refers to the use of the composition of the present invention for plant disease control purposes.

[0060] 2-2. Composition 2-2-1. Components The composition of the present invention contains a bacteriophage, which is a lytic agent as described in the first embodiment, as an essential component. It may also contain an agriculturally acceptable carrier and / or medium, provided that it does not inhibit or suppress the lytic activity of the phage against target bacteria. Furthermore, it may contain other active ingredients as needed. Each component will be described in detail below.

[0061] (1) Active ingredient (bacteriolytic agent) The composition of the present invention includes the bacteriolytic agent described in the first embodiment as an essential active ingredient. In the plant disease control composition, the target bacteria of the present invention are lysed by this active ingredient, and thus plant diseases caused by the target bacteria can be prevented or treated.

[0062] The specific composition of the lysing agent is described in detail in the first embodiment, so it will not be explained here.

[0063] The amount of active ingredient contained per unit volume in the composition depends on various conditions such as the dosage form, the type of plant pathogenic bacteria, the type of target plant, the application site, and the application method when used for plant disease control. It is preferable that the active ingredient, the phage, contains a sufficient amount to come into contact with and infect plant pathogenic bacteria that have infected the target plant. Therefore, within the scope of common technical knowledge in the field, the amount of lytic agent contained in the plant disease control composition of the present invention should be determined by considering each condition so that it becomes an effective amount against the target bacteria after application.

[0064] (2) Agriculturally acceptable carriers and media "Agriculturally acceptable carriers and / or media" means substances that facilitate the application of a composition, maintain the viability and infectivity of the phage, which is the active ingredient, and / or control the rate of action, and that have no or very little harmful effects on the environment such as soil and water quality when applied in the field, and that have no or very little harmful effects on animals, especially humans.

[0065] (2-1) Carriers Specific examples of agriculturally acceptable carriers include surfactants, protective agents, and excipients. If desired, small amounts of wetting agents, emulsifiers, and pH buffers may also be used. The carrier may be added in advance or added immediately before application.

[0066] Surfactants have the effect of improving the physicochemical properties of a composition with respect to plant tissue, such as wettability, emulsification, dispersibility, penetration, adhesion, defoaming, and spreading properties. Surfactants can be used as the main component of pesticide adjuvants called spreading agents. Examples of spreading agents include nonionic surfactants, combinations of nonionic and anionic surfactants, paraffinic surfactants, and paroxyethylene resin acid esters. More specifically, examples include polyoxyethylene alkyl ether compounds, polyoxyethylene fatty acid ester compounds, lignin sulfonate compounds, naphthylmethanesulfonate compounds, alkyl sulfosuccinate compounds, and tetraalkylammonium salt compounds.

[0067] Protective agents are expected to have effects such as reducing damage caused by ultraviolet light in the case of phages. Examples include skim milk, casein, and gelatin.

[0068] Examples of excipients include glucose, lactose, sucrose, gelatin, starch, malt, and wheat flour.

[0069] (2-2) Solvents Specific examples of agriculturally acceptable solvents include water (including aqueous solutions), buffers, or liquid culture media. The solvent is preferably a sterile liquid.

[0070] (3) Other active ingredients In addition to the lytic agent described in the first embodiment, the composition of the present invention may contain one or more other active ingredients having the same and / or different pharmacological effects, to the extent that they do not affect the lytic activity of the phages constituting the lytic agent.

[0071] Other active ingredients may be of any kind. For example, they may be phages that have lytic activity against the same and / or different bacteria. Examples of phages that have lytic activity against the same bacteria include other phages that specifically recognize and bind to Xanthomonas bacteria, similar to the phages that constitute the lytic agent described in the first embodiment. For example, even if the target bacteria are the same, if the phages recognize different cell surface receptors, a synergistic or complementary effect of lytic activity can be expected depending on the combination.

[0072] When the composition of the present invention contains multiple types of phages, the specific types of phages are not particularly limited. For example, only the phages described herein may be included as active ingredients, or any other phages may be included as additional active ingredients. Furthermore, phages with similar host ranges and / or phages with dissimilar host ranges may be included. Examples of combinations of phages with similar host ranges include combinations that commonly exhibit lytic activity against bacteria of the same genus (e.g., Xanthomonas), combinations of phages that commonly exhibit lytic activity against one or more species of bacteria of the same genus (e.g., Xanthomonas arboricola), and combinations of phages that commonly exhibit lytic activity against one or more pathogenic types of bacteria of the same genus (e.g., Xanthomonas arboricola pv. pruni). Furthermore, examples of phage combinations with dissimilar host ranges include combinations of phages that exhibit lytic activity against bacteria belonging to a specific genus (e.g., Xanthomonas) and those that do not, combinations of phages that exhibit lytic activity against a specific pathogenic type of bacteria in a specific genus (e.g., Xanthomonas arboricola pv. pruni) and those that do not, combinations of phages that exhibit lytic activity against a specific species of bacteria in a specific genus (e.g., Xanthomonas citri subsp. citri) and those that do not, and combinations of phages that specifically exhibit lytic activity against bacteria of a specific genus (e.g., Xanthomonas), species (e.g., Xanthomonas citri), or pathogenic type (e.g., Xanthomonas arboricola pv. pruni) and those that exhibit lytic activity nonspecifically.

[0073] For example, the composition of the present invention may contain a combination of two or more phages from the first to third phages described herein. Specifically, for example, it may contain a combination of two or more phages selected from the group consisting of the first phage, the second phage, and the third phage.

[0074] The number of first to third phages described herein included in the composition of the present invention is not particularly limited, as long as there is one or more types. For example, it may include two or more types or three or more types of phages. Furthermore, the composition of the present invention may include multiple types of phages whose genomic DNA sequences differ from each other, even though they fall within the range of each phage (e.g., the first phage). Specifically, it may include two types of first phages.

[0075] The plant disease control composition of the present invention may, in addition to the lytic agent described in the first embodiment, contain as an active ingredient one or more other phages that specifically recognize the Xanthomonas bacteria described above and have lytic activity.

[0076] The types of other phages that specifically recognize Xanthomonas bacteria and possess lytic activity are not particularly limited. Specific examples include the phages, their variants, and modified forms disclosed in WO 2023 / 191074 and / or WO 2024 / 248029.

[0077] For example, phages that contain in their genomic DNA a gene encoding the amino acid sequence shown in Sequence ID No. 1 of WO 2023 / 191074 as a common amino acid sequence for tail fiber proteins, specifically phages that contain in their genomic DNA a gene encoding the amino acid sequence shown in any of Sequence IDs No. 2 to 4 of WO 2023 / 191074, and phages that contain in their genomic DNA a gene encoding an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in any of Sequence IDs No. 1 to 4 of WO 2023 / 191074, or an amino acid sequence having 90% or more sequence identity. Specific examples of genes encoding such amino acid sequences include genes consisting of the nucleotide sequence shown in any of Sequence IDs No. 5 to 7 of WO 2023 / 191074, and nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in any of Sequence IDs No. 5 to 7 of WO 2023 / 191074, or nucleotide sequences having 90% or more sequence identity. Examples of genomic DNA of phages containing such genes include genomic DNA consisting of the nucleotide sequence shown in any of the sequence numbers 8 to 10 of WO 2023 / 191074, and genomic DNA consisting of a nucleotide sequence in which one or more bases are added, deleted, and / or substituted in the nucleotide sequence shown in any of the sequence numbers 8 to 10 of WO 2023 / 191074, a nucleotide sequence having 90% or more sequence identity, a nucleotide sequence in which one or more bases are added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence encoding the amino acid sequence shown in any of the sequence numbers 2 to 4 of WO 2023 / 191074, or a genomic DNA consisting of a nucleotide sequence in which a nucleotide sequence other than the nucleotide sequence encoding the amino acid sequence shown in any of the sequence numbers 2 to 4 of WO 2023 / 191074 has 80% or more sequence identity.

[0078] Furthermore, examples include phages whose genomic DNA contains a gene encoding the amino acid sequence shown in Sequence ID No. 11 or 45 of WO 2023 / 191074 as the amino acid sequence of the tail fiber protein, and phages whose genomic DNA contains a gene encoding an amino acid sequence in which one or more amino acids other than those at positions 278 and 350 have been added, deleted, and / or substituted, or an amino acid sequence having 90% or more sequence identity. Specific examples of genes encoding such amino acid sequences include genes consisting of the nucleotide sequence shown in Sequence ID No. 12 or 46 of WO 2023 / 191074, and nucleotide sequences in which one or more nucleotides have been added, deleted, and / or substituted, or nucleotide sequences having 90% or more sequence identity. Furthermore, specific examples of genomic DNA for phages containing such genes include, for example, genomic DNA consisting of the nucleotide sequence shown in Sequence ID No. 13, 47, or 48 of WO 2023 / 191074, and genomic DNA consisting of a nucleotide sequence in which one or more bases are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 13, 47, or 48 of WO 2023 / 191074, a nucleotide sequence having 90% or more sequence identity, a nucleotide sequence in which one or more bases are added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence encoding the amino acid sequence shown in Sequence ID No. 11 or 45 of WO 2023 / 191074, or a genomic DNA consisting of a nucleotide sequence in which a nucleotide sequence other than the nucleotide sequence encoding the amino acid sequence shown in Sequence ID No. 11 or 45 of WO 2023 / 191074 has 80% or more sequence identity.

[0079] Furthermore, examples include genomic DNA containing the nucleotide sequence shown in any of sequence numbers 14, 18, 19, and 32-36 of WO 2023 / 191074 and sequence numbers 1 and 3 of WO 2024 / 248029; genomic DNA containing the nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in any of sequence numbers 14, 18, 19, and 32-36 of WO 2023 / 191074 and sequence numbers 1 and 3 of WO 2024 / 248029; or phages having genomic DNA containing the nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in any of sequence numbers 14, 18, 19, and 32-36 of WO 2023 / 191074 and sequence numbers 1 and 3 of WO 2024 / 248029.

[0080] Furthermore, examples include phages whose genomic DNA contains a gene encoding the amino acid sequence shown in Sequence ID No. 15 or 42 of WO 2023 / 191074 as the amino acid sequence of the tail fiber protein, and phages whose genomic DNA contains a gene encoding an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 15 or 42 of WO 2023 / 191074, or an amino acid sequence having 90% or more sequence identity. Specific examples of genes encoding such amino acid sequences include genes consisting of the nucleotide sequence shown in Sequence ID No. 16 or 43 of WO 2023 / 191074, and nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 16 or 43 of WO 2023 / 191074, or nucleotide sequences having 90% or more sequence identity. Furthermore, specific examples of genomic DNA for phages containing such genes include, for example, genomic DNA consisting of the nucleotide sequence shown in Sequence ID No. 17 or 44 of WO 2023 / 191074, and nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 17 or 44 of WO 2023 / 191074, nucleotide sequences having 90% or more sequence identity, nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in nucleotide sequences other than the nucleotide sequence encoding the amino acid sequence shown in Sequence ID No. 15 or 42 of WO 2023 / 191074, or genomic DNA consisting of nucleotide sequences in which nucleotide sequences other than the nucleotide sequence encoding the amino acid sequence shown in Sequence ID No. 15 or 42 of WO 2023 / 191074 have 80% or more sequence identity.

[0081] Furthermore, examples include phages containing in their genomic DNA a gene encoding the amino acid sequence shown in Sequence ID No. 21 of WO 2023 / 191074, and phages containing in their genomic DNA a gene encoding an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 21 of WO 2023 / 191074, or an amino acid sequence having 90% or more sequence identity. Specific examples of genes encoding such amino acid sequences include genes consisting of the nucleotide sequence shown in Sequence ID No. 22 of WO 2023 / 191074, and nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 22 of WO 2023 / 191074, or nucleotide sequences having 90% or more sequence identity. Furthermore, specific examples of genomic DNA for phages containing such genes include, for example, genomic DNA consisting of the nucleotide sequence shown in Sequence ID No. 23 of WO 2023 / 191074, nucleotide sequences in which one or more bases are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 23 of WO 2023 / 191074, nucleotide sequences having 90% or more sequence identity, nucleotide sequences in which one or more bases are added, deleted, and / or substituted in nucleotide sequences other than the nucleotide sequence encoding the amino acid sequence shown in Sequence ID No. 21 of WO 2023 / 191074, or genomic DNA consisting of nucleotide sequences in which nucleotide sequences other than the nucleotide sequence encoding the amino acid sequence shown in Sequence ID No. 21 of WO 2023 / 191074 have 80% or more sequence identity.

[0082] Furthermore, examples include phages containing in their genomic DNA the TTPA (Tail-Tubular protein A) gene consisting of the amino acid sequence shown in Sequence ID No. 24, 49, 60, or 37 of WO 2023 / 191074 and the TTPB (Tail-Tubular protein B) gene consisting of the amino acid sequence shown in Sequence ID No. 57, 50, 61, or 38, 54, or 59, and phages containing in their genomic DNA the TTPA gene encoding an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 24, 49, or 37, or 57, 50, or 38, 54, or 59 of WO 2023 / 191074, or an amino acid sequence having 90% or more sequence identity. Specific examples of genes encoding such amino acid sequences include genes consisting of the nucleotide sequence shown in sequence numbers 26 or 39, or 58, 40 or 55 of WO 2023 / 191074, and nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in sequence numbers 26 or 39, or 58, 40 or 55 of WO 2023 / 191074, or nucleotide sequences having 90% or more sequence identity. Furthermore, specific genomic DNA of phages containing such genes include, for example, genomic DNA consisting of the nucleotide sequence shown in any of sequence numbers 28, 29, 41, or 56 of WO 2023 / 191074, and nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in any of sequence numbers 28, 29, 41, or 56 of WO 2023 / 191074, nucleotide sequences having 90% or more sequence identity, nucleotide sequences in which one or more nucleotides are added, deleted, and / or substituted in nucleotide sequences other than those encoding the amino acid sequences shown in sequence numbers 24 or 37, or 57, 50, 38, 54, or 59 of WO 2023 / 191074, or WO Examples include genomic DNA consisting of nucleotide sequences having 80% or more sequence identity with nucleotide sequences other than those encoding the amino acid sequences shown in Sequence ID No. 24 or 37, or 57, 50 or 38, 54 or 59 of 2023 / 191074.

[0083] Furthermore, examples include genomic DNA having a total length of 40,000 bases or less and containing the nucleotide sequence shown in Sequence ID No. 2 of WO 2024 / 248029, and a total length of 40,000 bases or less; genomic DNA having a total length of 40,000 bases or less and containing a nucleotide sequence in which one or more bases are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 2 of WO 2024 / 248029; or genomic DNA having a total length of 40,000 bases or less and containing a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2 of WO 2024 / 248029.

[0084] Sequence identity as used herein is not particularly limited. Specifically, for example, relative to a reference sequence, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90.0% or more, 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% The above may indicate sequence identity of 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0085] In addition, it is preferable that the sequence identity of the amino acid sequence be 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. As already stated, in this specification, "multiple" means 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0086] Other active ingredients may include insecticides, herbicides, fertilizers (e.g., urea, ammonium nitrate, superphosphates), and, if necessary, known chemical pesticides, antibiotics, and biopesticides.

[0087] 2-2-2. Dosage Form The composition of the present invention may be in any dosage form when used as a plant disease control composition, as long as it can maintain the infection site of the target bacteria on the target plant, its ability to settle on the target plant, and / or the ease with which the phage, which is the active ingredient, infects the target bacteria. For example, the plant disease control composition may be a liquid formulation or wettable powder formulation in which it is suspended in a suitable solution, or it may be a solid formulation such as a powder, granules, or gel formulation in which it is mixed with a carrier and solidified. For example, when the infection site of the target bacteria on the target plant is the above-ground leaves, flowers, fruits, stems, branches, or trunk, a liquid formulation, wettable powder, or gel formulation is preferred, although not limited to these, as it can spread widely to these infection sites and has high settlement ability. On the other hand, when the infection site of the target bacteria is the underground roots or rhizomes, a powder formulation or granules is preferred, although not limited to these, as it can be slowly released in the soil and exert a sustained effect on the infection site.

[0088] 2-3. Method of Application When the composition of the present invention is used as a plant disease control composition, any method known in the art that allows the plant disease control composition to be applied to the target plant is acceptable and is not particularly limited. Since the phage, which is the active ingredient of the plant disease control composition, can penetrate from the entire surface of the plant, such as the stems, leaves, and roots of the target plant, it can be applied in an appropriate manner according to the purpose. For example, if the application site is the above-ground part such as the stems and leaves, the plant disease control composition should be applied so that it comes into direct contact with the application site. Examples of direct contact include coating, spraying, scattering, or immersing the application site of the plant disease control composition. It is particularly preferable to apply the composition to the site of infection by target bacteria or to a site that is at risk of infection. If the application site is the underground part such as the roots, it can be applied indirectly by adding it to the soil, or if it is a growing medium, by adding it to the growing medium. The term "soil" here is not particularly limited as long as it is soil in which the target plant can grow. Typically, planting soil containing appropriate nutrients and having an appropriate pH value is used. The location of the soil is not important. Furthermore, "culture medium" refers to an artificially prepared culture medium for the target plant. It may be a solid medium such as agar medium or a liquid medium. Examples of culture media include isolation beds, root-restricted pots, or seedbeds. The composition of the culture medium may be any known culture medium composition in the field. It can be appropriately selected depending on the type of plant, etc.

[0089] The phage of the present invention has high freeze stability. Therefore, it can be cryopreserved as appropriate. The storage conditions are not particularly limited, but for example, it may be stored using a cryopreservation solution and / or host bacteria, or the phage may simply be suspended in an aqueous solution such as a buffer and stored. The freezing temperature is not particularly limited as long as it is 0°C or lower, but for example, the upper limit can be 0°C, -5°C, -10°C, -15°C, -18°C, -20°C, etc., and the lower limit can be -196°C, -150°C, -100°C, -80°C, -78°C, -75°C, -50°C, -40°C, -30°C, -25°C, -20°C, etc. Furthermore, it can be frozen at temperatures in the following ranges, for example: -196°C to 0°C, -150°C to -10°C, -78°C to -10°C, -75°C to -10°C, -50°C to -15°C, -40°C to -15°C, -30°C to 0°C, -30°C to -10°C, -30°C to -15°C, -30°C to -18°C, -25°C to 0°C, -25°C to -10°C, -25°C to -15°C, -25°C to -18°C, -20°C to 0°C, -20°C to -5°C, -20°C to -10°C, etc.

[0090] There are no specific limitations on the storage period, but it can be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, or 10 days or more. Alternatively, the storage period can be, for example, 1 year or less, 6 months or less, 3 months or less, 2 months or less, 1 month or less, 20 days or less, 15 days or less, 10 days or less, etc.

[0091] 2-4. Target Plants The target plants of the plant disease control composition of the present invention are not particularly limited in type, as long as they are plants that can develop plant diseases caused by the target bacteria of the present invention. They may be either angiosperms or gymnosperms. Furthermore, they may be herbaceous plants or woody plants. Suitable specific examples of target plants include agriculturally important plants, such as crop plants such as grains, vegetables, and fruits, and ornamental plants. Specifically, among monocots, examples include grasses (e.g., rice, wheat, barley, maize, sugarcane, sorghum, sorghum, turfgrass), bananas (e.g., bananas), onions (e.g., leeks, onions, garlic, chives), and lilies (e.g., lilies, tulips). Furthermore, dicotyledonous plants include Brassicaceae plants (e.g., cabbage, broccoli, cauliflower, radish, Chinese cabbage, rapeseed), Asteraceae plants (e.g., lettuce, burdock, chrysanthemum), Juglandaceae plants (e.g., walnut, Japanese walnut, black walnut, pecan (pecan nut)), Fabaceae plants (e.g., soybean, peanut, pea, green bean, lentil, chickpea, broad bean, licorice), Solanaceae plants (e.g., tomato, eggplant, potato, tobacco, bell pepper, chili pepper, petunia, shishito pepper), and Rosaceae plants (e.g., strawberry, apple). This includes pears, peaches, loquats, almonds, plums, roses, apricots, cherries), Cucurbitaceae plants (e.g., cucumbers, melons, pumpkins, melons, watermelons), Anacardiaceae plants (e.g., mangoes, pistachios, cashews), Lauraceae plants (e.g., avocados), Rutaceae plants (e.g., mandarins, summer oranges, Iyokan oranges, oranges, sweet oranges, grapefruits, lemons, yuzu), Convolvulaceae plants (e.g., sweet potatoes), Theaceae plants (e.g., tea plants), and Vitaceae plants (e.g., grapes).

[0092] 2-5. Target Plant Diseases The target plant diseases for which the plant disease control composition of the present invention is applied include all plant diseases caused by the target bacteria of the present invention. Preferably, these are plant diseases caused by bacteria of the genus Xanthomonas. Examples include bacterial spot seen in peaches, bacterial blight seen in walnuts, bacterial pustule seen in soybeans, angular leaf spot seen in strawberries, bacterial blight seen in tomatoes, bell peppers and lettuce, black rot seen in cabbage, Chinese cabbage and broccoli, bacterial canker seen in oranges and grapefruits, angular leaf spot seen in cotton, and leaf blight seen in rice.

[0093] 2-6. Composition The present invention further provides a composition comprising the lytic agent described in the first embodiment as an active ingredient. The composition only needs to contain the lytic agent described in the first embodiment and have lytic activity, and its use is not particularly limited. For example, it can be a lytic composition, a composition for lytic of Xanthomonas bacteria, and so on.

[0094] The composition, application method, and target plants of these compositions shall be in accordance with the details described above in this embodiment. Even if the compositions are used for purposes other than plant disease control, they may secondarily exhibit control effects against the various diseases described above as target plant diseases.

[0095] 3. Method for Controlling Plant Diseases 3-1. Overview The third aspect of the present invention is a method for controlling plant diseases. The method for controlling plant diseases of the present invention is characterized by controlling plant diseases of a target plant by applying the plant disease control composition described in the second aspect to the target plant.

[0096] According to the plant disease control method of the present invention, it is possible to control bacterial plant diseases, particularly those caused by bacteria of the genus Xanthomonas, in target plants.

[0097] 3-2. Method The plant disease control method of the present invention includes a contact step as an essential step.

[0098] The "contact step" is the step of bringing the plant disease control composition described in the second embodiment into contact with the target plant. This step basically follows "2-3. Application method" in the plant disease control composition of the second embodiment.

[0099] In this embodiment, "contact" means that the plant disease control composition comes into contact with the target plant. More specifically, it means that the lysing agent described in the first embodiment, i.e., the phage, which is the active ingredient of the plant disease control composition, comes into contact with the plant body of the target plant, preferably with the site of infection by the target bacteria or a site that is at risk of infection. This step aims to infect the target bacteria with the phage, which is the active ingredient, thereby lysing the target bacteria. As a result, a control effect against plant diseases caused by the target bacteria can be achieved.

[0100] Contact may be either direct or indirect. In this embodiment, direct contact means that the plant disease control composition comes into direct contact with a predetermined part of the target plant. Specifically, this means, for example, applying, spraying, scattering, or immersing a liquid or gel-like plant disease control composition on the plant body of the target plant. In this case, the plant body to be contacted is mainly the leaves, flowers, fruits, stems, branches, and / or trunk. On the other hand, in this embodiment, indirect contact means that the plant disease control composition comes into contact with a predetermined part of the target plant via an intermediary. For example, this means applying a granular plant disease control composition to the soil around the roots of the target plant. The active ingredient, phage, is transported via water in the soil and eventually absorbed by the roots.

[0101] 3-3. Effects The phage, the active ingredient of the composition obtained by the present invention, can efficiently kill target bacteria, thus contributing to the prevention and suppression of diseases caused by target bacteria. Furthermore, the detection and identification of target bacteria based on its specific lytic activity enables the diagnosis of diseases. Copper compounds and antibiotics have been conventionally used as agents against Xanthomonas bacteria, but these agents can cause pharmacotoxicity and disrupt the balance of the bacterial flora. For example, some strains of Pseudomonas fluorescens have been shown to have a plant growth promoting effect (Haas D., Defago G., Nature Reviews in Microbiology, 2005, 3(4), 307-19), and copper compounds and antibiotics are highly likely to kill bacteria that have a symbiotic relationship with such plants. On the other hand, since phages are biologically derived substances, no pharmacotoxicity has been reported, and their high specificity means that their impact on the balance of the bacterial flora is extremely limited.

[0102] 4. Method for Identifying Xanthomonas Bacteria 4-1. Overview The fourth aspect of the present invention is a method for identifying Xanthomonas bacteria. The identification method of the present invention is characterized by identifying Xanthomonas bacteria by utilizing the host specificity of the phage constituting the lytic agent described in the first aspect.

[0103] According to the present invention, it is possible to determine and identify whether an unidentified plant pathogenic bacterium that caused a plant disease is a bacterium of the genus Xanthomonas.

[0104] 4-2. Method The identification method of the present invention includes a culture step, a mixing step, a mixture culture step, and a determination step as essential steps, and an isolation step as an optional step. Each step will be described below.

[0105] (1) Isolation Process The "isolation process" is the process of isolating the test bacteria from plant tissue affected by plant disease. This process is selective and may be performed only if necessary.

[0106] "Test bacterium" refers to a plant pathogenic bacterium used in the method for identifying Xanthomonas bacteria according to the fourth aspect of the present invention, whose species has not been identified.

[0107] The plant tissue can be from any part of a plant that has developed a plant disease, but parts where the symptoms of the plant disease are clearly visible are preferred. For example, in the case of a peach tree that has developed bacterial spot disease, the leaves showing the disease should be used.

[0108] To isolate the test bacteria from collected plant tissue, the diseased specimen can be immersed in a solvent such as water for extraction; the specimen may be fragmented or crushed during extraction. Subsequently, the extract can be streaked onto an agar plate, and single colonies can be picked.

[0109] (2) Culturing process The "culturing process" is the process of culturing the isolated test bacteria to obtain a culture. The method for culturing the test bacteria may be any method known in the field.

[0110] A "culture" is a substance obtained by culturing the test bacteria, and may be either a liquid or a solid.

[0111] In this process, since the test bacteria are unidentified, it is desirable to use a culture medium capable of broadly culturing plant pathogenic bacteria. At a minimum, a culture medium capable of culturing Xanthomonas bacteria, which are the target bacteria of this invention, should be used. Such a culture medium may contain one or more components selected from, for example, protein enzyme hydrolysates such as peptone and tryptone, biological extracts such as potato dextrose and yeast extract, amino acids such as glutamic acid or their salts, sugars such as glucose and sucrose, and inorganic salts such as sodium chloride, magnesium chloride, and potassium dihydrogen phosphate. Specific culture media and compositions include LB medium (tryptone, yeast extract, sodium chloride), YPG medium (yeast extract, peptone, glucose), PD medium (potato dextrose), and Suwa medium with added peptone (sucrose, glutamic acid, peptone).

[0112] The isolated test bacteria are seeded in the culture medium and cultured under appropriate conditions. Culture conditions include, for example, 20-40°C, 20-30°C, 22-28°C, or 24-26°C, and the culture can be obtained by culturing while stirring. The culturing time is not limited, but it is sufficient to cultivate until, for example, the turbidity at a wavelength of 600 nm reaches approximately 1.0. This step yields a culture solution of the test bacteria. Furthermore, the culture may be carried out in two or more stages. For example, after culturing in a liquid medium, a liquid medium containing soft agar can be added to the culture solution, which can then be poured onto a solid medium such as an agar plate, solidified, and further cultured.

[0113] (3) Mixing step The "mixing step" is a step of mixing the culture obtained in the cultivation step with the lytic agent described in the first embodiment to obtain a mixture.

[0114] A "mixture" is a mixture of the culture and the lysant, and may be in liquid or solid form.

[0115] The method of mixing the culture and the lysant is not particularly limited, as long as they can be mixed. The lysant described in the first embodiment may be in solid form, but may also be administered in liquid form suspended in water or a liquid culture medium.

[0116] If both the culture and the lysant are liquids, the volume ratio of the culture to the lysant can be 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1. After administration, the culture and lysant should be thoroughly mixed by stirring or other means. On the other hand, if a liquid medium containing soft agar is layered as described above, the culture is solid. In this case, the lysant may be dropped onto the solid culture, such as the gel surface, to mix the two on the solid medium and obtain a mixture.

[0117] (4) Mixture culture step The "mixture culture step" is a step of culturing the mixture under predetermined conditions.

[0118] Furthermore, when culturing the mixture, it is also possible to add a liquid medium containing soft agar to the mixture, pour it onto a solid medium such as agar plate, allow it to solidify, and then cultivate it further.

[0119] The basic procedure for this step is the same as that for the culture step described above. In this step, although not limited to this, it is preferable to perform culture based on a so-called plaque assay method so that it is easier to confirm whether or not the test bacteria have been lysed by the phages constituting the lytic agent in the determination step described below. For example, a portion of the mixture can be mixed with a soft agar medium of the same composition, and then, before the soft agar medium solidifies, it can be poured onto an agar medium of the same composition and spread over the entire medium. After that, it can be cultured under the same conditions as in the culture step described above.

[0120] (5) Determination step The "determination step" is a step in which, if the test bacteria after the culture step are lysed, the test bacteria are determined to be of the genus Xanthomonas.

[0121] While there are no limitations on how to determine whether lysis has occurred, for example, if using a plaque assay method, the determination can be made based on the presence or absence of plaque formation. If plaque is present on the soft agar medium that has been spread and solidified on the agar plate after the aforementioned mixture culture step, it indicates that the test bacteria have been lysed by infection with the phages constituting the lytic agent of the present invention. Therefore, the test bacteria at this time can be determined to be Xanthomonas bacteria. On the other hand, if the test bacteria have grown throughout the agar plate and no plaque is present at all, the test bacteria can be determined not to be Xanthomonas bacteria.

[0122] To obtain a more accurate determination, a negative control may be prepared simultaneously in the mixed culture step, which is mixed with a culture medium that does not contain a lysating agent, and / or a positive control in which identified Xanthomonas bacteria are used from the culture step instead of the test bacteria. It may be confirmed that no plaques are formed in the negative control and that plaques are observed in the positive control.

[0123] 4-3. Effects: The method for identifying Xanthomonas bacteria of the present invention makes it possible to identify whether or not Xanthomonas bacteria are the cause of plant diseases.

[0124] Furthermore, according to the Xanthomonas bacterium identification method of the present invention, it is possible to detect whether or not Xanthomonas bacterium is present in the lesions of plants that have developed plant diseases suspected to be caused by Xanthomonas bacterium.

[0125] <Example 1: Isolation of a Novel Bacteriophage and its Lysolytic Activity (1)> (Objective) To isolate a novel bacteriophage that has lytic activity against plant pathogenic bacteria and to verify its lytic activity against plant pathogenic bacteria.

[0126] (Methods and Results) (1) Acquisition and Cultivation of Plant Pathogenic Bacteria In this example, all plant pathogenic bacteria were obtained from the National Agriculture and Food Research Organization (NARO). Table 2 lists each bacterium used in this example along with its NARO deposit numbering.

[0127]

[0128] For control purposes, a strain of Pseudomonas fluorescens, which has been reported to have plant growth-promoting effects and other environmental benefits, was obtained from the NCIMB (National Microbial Identification Bureau), a laboratory within the UK National Microbial Conservation Center (UKNCC) (NCIMB-ID: 10460).

[0129] For culturing Xanthomonas bacteria and Pseudomonas fluorescens species, a liquid medium (YPG Broth) was used, prepared by dissolving 1 g of peptone, 1 g of yeast extract, and 2 g of glucose in 1 L of H2O and autoclaving it. As an agar medium, an agar medium (referred to as "YPG Agar" when using YPG Broth) was prepared by adding 15 g of agar per L of the above Broth (YPG Broth) and autoclaving it. Furthermore, a soft agar medium (Top Agar) was prepared by adding 5 g of agarose per L of the above Broth, autoclaving the Top Agar, storing it at approximately 50°C, and using it as needed.

[0130] Each of the above bacterial strains, delivered in a dry powder state, was suspended in 0.1 mL of broth, then streaked in agar (YPG agar) at 25°C to isolate single colonies. The isolated colonies were inoculated into broth and cultured with shaking at 25°C to serve as the pre-culture medium. For the main culture, the pre-culture medium was inoculated into broth and cultured at 25°C for 10 to 30 hours until the turbidity (Optical Density 600 nm) reached approximately 1.0. The culture medium after incubation was used directly as the bacterial suspension.

[0131] (2) Isolation and Purification of the First Phage The novel phages were isolated from natural wastewater or soil obtained in Japan. The phage isolation method was based on a standard plaque assay method. First, wastewater from ponds or lakes, or wastewater obtained by suspending soil in water, was filtered through a 0.45 μm filter to prepare a phage-containing solution. Next, equal volumes of bacterial suspension and phage-containing solution were mixed and left at room temperature for about 10 minutes. Then, 0.2 mL of the bacterial / phage mixture was added to 3 mL of Top Agar, quickly mixed using a vortex mixer, and then poured onto the Agar. After the Top Agar solidified, it was incubated at 25°C for about 12 hours. A lytic plaque was formed on the bacterial lawn formed by the culture. Subsequently, the gel from the plaque portion was aspirated using a tip-cutting device, and phages with lytic activity against Xanthomonas bacteria were isolated. Subsequently, the phage was purified by repeating this procedure using a phage-containing solution with a high concentration of the isolated phages as a substitute for wastewater.

[0132] Using a method that detects lytic plaques formed on soft agar plates amplified with any of the above bacterial strains, a total of two new phages were isolated from natural wastewater and soil. The two new phages isolated in Example 1 will be referred to as "Phage 1".

[0133] The isolated phages were suspended in SM Buffer and collected as a phage-containing solution after passing through a 0.2 μm filter. This phage-containing solution was mixed with the bacterial suspension under the conditions described above, and the phages were isolated again. This procedure was repeated several times to further purify the phages. The composition of the SM Buffer is shown in Table 3.

[0134]

[0135] (3) Amplification and Purification of the First Phage The first phage, which was isolated and purified, was amplified and purified using the plate lysate (PL) method, which is an amplification method using a plaque assay. A bacterial / phage mixture was prepared so that many plaques would form on the agar, and after mixing with Top Agar, it was spread on YPG Agar and cultured. Then, 3 mL of SM Buffer was added to the Top Agar on which the plaques had formed, and the mixture was shaken at 25°C for about 30 minutes. The supernatant was passed through a 0.2 μm filter to collect the recovered solution containing the phage.

[0136] For the purification of the first phage, 1 g of PEG 6000 (final concentration 10%) and 0.4 g of NaCl (final concentration 4%) were added to 10 mL of the recovered solution and dissolved. The mixture was then rotated overnight at 4°C using a rotator. After that, it was centrifuged at ×15,000 g / 4°C / 60 minutes, and the supernatant was removed. The recovered pellet was resuspended in 0.5 mL of SM Buffer. Subsequently, 0.5 mL of chloroform was added, the mixture was vigorously stirred, and left on ice for 6 hours. After centrifuging at ×8,000 g / 4°C / 10 minutes, the upper layer was carefully collected to obtain the phage purified solution. The concentration of the phage purified solution is generally expressed as titer [PFU / mL] based on the number of plaques (Plaque Forming Units, PFU) obtained by the plaque assay method, and serves as one indicator of lytic activity. The titer of the first phage purified solution prepared was determined by a plaque assay against Xanthomonas arboricola pv. pruni (MAFF No. 311351) using a solution diluted as appropriate, and 10 8 We confirmed that the level was PFU / mL or higher.

[0137] (4) Evaluation of the host range of the first phage The host range of the first phage was evaluated using the spot test method. 0.1 mL of bacterial suspension was added to 3 mL of Top Agar and mixed, then poured onto the Agar and spread across the entire plate to solidify. In addition to the bacterial suspensions of each Xanthomonas species shown in Table 2 prepared in (1) above, a control suspension of Pseudomonas fluorescens was also prepared. Then, approximately 5 μL of phage purified solution was dropped onto the plate and incubated at 25°C for approximately 12 hours. If a circular area (approximately 1 cm in diameter) became clear at the drop location on the plate where the bacterial lawn was formed, it was determined that the dropped phage had lytic activity against that strain.

[0138] An example of the results is shown in Figure 1. When the first phage exhibits lytic activity, only the area where the phage purified solution is dropped into the bacterial lawn formed on the plate becomes clear. All of the first phages obtained by this invention exhibited lytic activity against all strains belonging to the three bacterial species shown in Table 2: Xanthomonas arboricola (including pv. pruni), Xanthomonas campestris (including pv. campestris and pv. vesicatoria), and Xanthomonas citri. On the other hand, it was confirmed that they did not exhibit lytic activity against Pseudomonas fluorescens. This suggests that the isolated first phage exhibits broad lytic activity against Xanthomonas bacteria.

[0139] Furthermore, this result demonstrates the potential of the first phage to be applicable to various plant diseases. For example, it is suggested to be useful in controlling bacterial spot disease in peaches, black rot in broccoli, and bacterial leaf spot in tomatoes, which are caused by Xanthomonas bacteria, and is expected to have high industrial value.

[0140] (5) Genome analysis of the first phage The genomic DNA sequence of the first phage was determined and analyzed.

[0141] (i) Preparation and sequencing of the genomic DNA of the first phage: TURBO DNA-free TMThe genome of the first phage was extracted using a kit (Thermo Fisher Scientific). Host bacterial genomic DNA, which would act as an impurity, was removed according to the kit's instructions. Subsequently, the phage outer shell molecules were degraded by Proteinase K treatment using NucleoSpin® Virus (Machery-Nagel) according to the instructions. After purifying the genomic DNA using a silica spin column, a genomic DNA solution of the first phage was prepared. Then, the concentration of genomic DNA was measured using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific), and 50 μL of the genomic DNA solution was prepared to a final concentration of 0.2 ng / μL. Next, the genome of the first phage was fragmented and adapter sequences were added by PCR using Nextera XT DNA Library Prep (Illumina) according to the instructions. Next, using the Agilent High Sensitivity DNA Kit (Agilent Technologies), electrophoresis was performed using a Bioanalyzer (Agilent Technologies) to measure the average bp size of the samples and determine the concentration of DNA fragments. Finally, using the Miseq Reagent kit (Illumina), samples for measurement were prepared according to the included manual, and measurements were performed using a next-generation sequencer, Miseq (Illumina). After preprocessing (trimming, etc.) of the obtained data using CLC genomics workbench (Qiagen), de novo assembly was performed to obtain contig sequences corresponding to the phage genome sequence.

[0142] (ii) Bioinformatics analysis based on genome sequence information Based on the base sequences of the genomic DNA of the first phage obtained in (i) above (SEQ ID NOs: 1 and 2), a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI-provided BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). When searching for known sequences with high sequence identity over a range of more than 90% of the total length (Query Cover), the sequence with the highest sequence identity score was the genomic DNA sequence of a phage targeting Xanthomonas bacteria called vB_Xar_IVIA-DoCa6 (access code: NC_073035.1), which had approximately 99.35% sequence identity over approximately 97% of the total length of SEQ ID NO: 1. In relation to SEQ ID NO: 2, it had approximately 99.2% sequence identity over approximately 96% of the total length of SEQ ID NO: 1.

[0143] Sequence identity is a numerical value relative to a range automatically aligned by the analysis server within the entire genome of the first phage. This range is displayed as the Query Cover. For example, the 99.35% sequence identity of vB_Pae_TR for sequence number 1 is calculated based on a region limited to 97% of the first phage's entire genome. Therefore, while calculating sequence identity for the entire genome is difficult, it is estimated to be at least lower than 99.35%, and typically equivalent to or lower than 97%.

[0144] Furthermore, sequence identity between the genomic DNA sequences of each phage (SEQ ID NO: 1 and 2) was analyzed using BLAST analysis with GENETYX-NGS, which is implemented in the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ). Sequence identity, using Average Nucleotide Identity (ANI) as an indicator, was 99.04% (ANI value) over 98.21% of the total length (comparison range). The comparison range (%) here refers to the percentage of the region in the genomic DNA sequence of the corresponding column that is aligned with the genomic DNA sequence of the corresponding row.

[0145] Although the target bacteria of the phage vB_Xar_IVIA-DoCa6, which has the genome sequence known above, are all Xanthomonas species, it has been reported that this phage's host range is such that it responds to Xanthomonas arboricola (pv. juglandis and pruni) but not to Xanthomonas campestris and Xanthomonas citri (Domingo-Calap ML et al., 2022, Microbiol Spectr 10:e02960-22). On the other hand, as mentioned above, the first phage obtained in this example showed lytic activity against Xanthomonas arboricola, as well as at least Xanthomonas campestris.

[0146] These results suggest that the first phage is a completely novel phage with a host range of the Xanthomonas genus that had not been previously reported.

[0147] <Example 2: Isolation of a Novel Bacteriophage and its Lysolytic Activity (2)> (Objective) To isolate a novel bacteriophage that has lytic activity against plant pathogenic bacteria and to verify its lytic activity against plant pathogenic bacteria.

[0148] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the target bacteria were plant pathogenic bacteria of the genus Xanthomonas, and the strains listed in Table 2 were used.

[0149] The cultivation of bacteria of the genus Xanthomonas and Pseudomonas fluorescens was carried out in accordance with the method described in "(1) Obtaining and culturing plant pathogenic bacteria" of Example 1.

[0150] (2) Isolation and Purification of the Second Phage The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were the same as those described in "(2) Isolation and Purification of the First Phage" of Example 1. As a result, one new type of phage was isolated from the natural wastewater and soil. This novel phage isolated in Example 2 will be referred to as the "second phage".

[0151] The isolated phages were suspended in SM Buffer and collected as a phage-containing solution after passing through a 0.2 μm filter. This phage-containing solution was mixed with the bacterial suspension under the conditions described above, and the phages were isolated again. The phages were purified by repeating this procedure several times.

[0152] (3) Amplification and Purification of the Second Phage The isolated and purified second phage was amplified and purified using the plate lysate (PL) method. The specific method was the same as described in "(3) Amplification and Purification of Phage" of Example 1. The titer of the prepared purified second phage solution was determined by a plaque assay using a solution diluted as appropriate, and 10 8 We confirmed that the level was PFU / mL or higher.

[0153] (4) Evaluation of the host range of the second phage The host range of the second phage was evaluated using the spot test method. The basic procedure was the same as the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0154] An example of the results is shown in Figure 2. When the second phage exhibits lytic activity, only the area where the phage purified solution is dropped into the bacterial lawn formed on the plate becomes clear. The second phage obtained by this invention showed lytic activity against all of the Xanthomonas species shown in Table 2. On the other hand, it did not show lytic activity against Pseudomonas fluorescens. This suggests that the isolated second phage exhibits broad lytic activity against Xanthomonas species.

[0155] The second phage, like the first phage, has a high potential to be applied to various plant diseases. These results suggest that it may be useful in controlling diseases caused by Xanthomonas bacteria, such as peach bacterial spot, broccoli black rot, and tomato bacterial leaf spot.

[0156] (5) Genome analysis of the second phage The genomic DNA sequence of the second phage was determined and analyzed.

[0157] (i) Preparation and sequencing of the genomic DNA of the second phage: TURBO DNA-free TMThe genome of the second phage was extracted using a kit (Thermo Fisher Scientific). The basic procedure followed the method described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of the genomic DNA of the first phage" in Example 1.

[0158] (ii) Bioinformatics analysis based on genome sequence information Based on the base sequence of the genomic DNA of the second phage obtained in (i) above (SEQ ID NO: 5), a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI-provided BLAST server. As a result of the search, a partial sequence of the genomic DNA of a phage targeting the genus Stenotrophomonas called vB_SmaS-DLP_6 (access code: KU682439.2) had the highest similarity score, exhibiting approximately 87.4% sequence identity over a range of approximately 97% of the full length of SEQ ID NO: 5. From this result, it is estimated that the sequence identity of SEQ ID NO: 5 relative to the full length corresponds to a value of approximately 84.7%. However, only bacteria of the genus Stenotrophomonas are known to exhibit lytic activity against the phage vB_SmaS-DLP_6 (Peters DL et al., PLoS One, 2017, 12(3)).

[0159] Generally, the characteristics related to phage host specificity and host range are thought to be largely determined by the role of tail fiber proteins (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16:760-773). Therefore, we searched for tail fiber genes from the genome sequence information of the aforementioned phage. Using the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ), we identified the nucleotide sequence shown in SEQ ID NO: 4, which is presumed to be the tail fiber gene, from the genome sequence of the second phage. As a result of searching for similar DNA sequences for this gene sequence, the tail fiber gene sequence of the aforementioned phage vB_SmaS-DLP_6 had the highest affinity score. The sequence identity of this nucleotide sequence was approximately 78.2% over 99% of the total length of the nucleotide sequence of SEQ ID NO: 4. Regarding the amino acid sequence of SEQ ID NO: 3, the tail fiber protein of vB_SmaS-DLP_6 also had the highest affinity score. The amino acid sequence identity was approximately 84.5% over approximately 99% of the total length. This indicates that the tail fiber protein of the second phage is novel, with no closely related proteins existing, and is a particularly characteristic protein of the phage of the present invention compared to known phages.

[0160] Based on these results, it was hypothesized that the second phage possesses a novel host range within the Xanthomonas genus that had not been previously reported, and that this characteristic is particularly driven by a novel tail fiber gene.

[0161] <Example 3: Isolation of a Novel Bacteriophage and its Lysolytic Activity (3)> (Objective) To isolate a novel bacteriophage that has lytic activity against plant pathogenic bacteria and to verify its lytic activity against plant pathogenic bacteria.

[0162] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the target bacteria were plant pathogenic bacteria of the genus Xanthomonas, and the strains listed in Table 2 were used.

[0163] The cultivation of bacteria of the genus Xanthomonas and Pseudomonas fluorescens was carried out in accordance with the method described in "(1) Obtaining and culturing plant pathogenic bacteria" of Example 1.

[0164] (2) Isolation and Purification of the Third Phage The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were the same as those described in "(2) Isolation and Purification of the First Phage" of Example 1. As a result, one new type of phage was isolated from the natural wastewater and soil. This novel phage isolated in Example 2 will be referred to as the "second phage".

[0165] The isolated phages were suspended in SM Buffer and collected as a phage-containing solution after passing through a 0.2 μm filter. This phage-containing solution was mixed with the bacterial suspension under the conditions described above, and the phages were isolated again. The phages were purified by repeating this procedure several times.

[0166] (3) Amplification and Purification of the Third Phage The plate lysate (PL) method was used to amplify and purify the isolated and purified third phage. The specific method was the same as described in "(3) Amplification and Purification of Phage" of Example 1. The titer of the prepared purified third phage solution was determined by a plaque assay using a solution diluted as appropriate, and 10 8 We confirmed that the level was PFU / mL or higher.

[0167] (4) Evaluation of the host range of the third phage The host range of the third phage was evaluated using the spot test method. The basic procedure was the same as the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0168] An example of the results is shown in Figure 3. When the third phage exhibits lytic activity, only the area where the phage purified solution is dropped into the bacterial lawn formed on the plate becomes clear. The third phage obtained by this invention showed lytic activity against all of the Xanthomonas species listed in Table 2. On the other hand, it did not show lytic activity against Pseudomonas fluorescens. This suggests that the isolated third phage exhibits broad lytic activity against Xanthomonas species.

[0169] The third phage, like the first phage, has a high potential to be applied to various plant diseases. These results suggest that it may be useful in controlling diseases caused by Xanthomonas bacteria, such as peach bacterial spot, broccoli black rot, and tomato bacterial leaf spot.

[0170] (5) Genome analysis of the third phage The genomic DNA sequence of the third phage was determined and analyzed.

[0171] (i) Preparation and sequencing of the genomic DNA of the third phage: TURBO DNA-free TM The genome of the second phage was extracted using a kit (Thermo Fisher Scientific). The basic procedure followed the method described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of the genomic DNA of the first phage" in Example 1.

[0172] (ii) Bioinformatics analysis based on genome sequence information Based on the base sequence of the genomic DNA of the third phage obtained in (i) above (SEQ ID NO: 6), a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI-provided BLAST server. As a result of the search, a partial sequence of the genomic DNA of a phage targeting the genus Xanthomonas called XAJ24 (access code: NC_047762.1) had the highest similarity score, exhibiting approximately 94.6% sequence identity over a range of about 90% of the full length of SEQ ID NO: 6. From this result, it is estimated that the sequence identity of SEQ ID NO: 6 relative to the full length corresponds to a value of approximately 85.1%.

[0173] Although the target bacteria of phage XAJ24, which possesses the genome sequence known above, are all Xanthomonas species, it has been reported that this phage responds to certain strains of Xanthomonas arboricola (pv. juglandis) and Xanthomonas campestris (pv. campestris) (Dora Domotor et al., Infection, Genetics and Evolution, 2016, Volume 43, Pages 371-377). This paper also evaluates responses to other Xanthomonas bacterial species, but there was no data or description indicating lytic activity against Xanthomonas citri.

[0174] These results suggest that the third phage is a completely novel phage with a host range of the Xanthomonas genus that had not been previously reported.

[0175] <Example 4: Further investigation of the lytic activity of novel bacteriophages> (Objective) To further investigate the lytic activity of the novel bacteriophages isolated in Examples 1-3.

[0176] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, all plant pathogenic bacteria were obtained from the National Agriculture and Food Research Organization (NARO). Table 4 lists each bacterium used in this example along with its NARO deposit numbering.

[0177]

[0178] (2) Phage amplification and purification Phages isolated and purified from natural wastewater or soil obtained in Japan by methods described in each of the literature, phages having the genomic DNA sequences of SEQ ID NOs. 10, 13, 14, 17, 19, 23, 29, 35, 41 and 44 in WO 2023 / 191074, and phages having the genomic DNA sequences of SEQ ID NOs. 1 to 3 in WO 2024 / 248029 were used as known phages. In addition, a phage having the genomic DNA sequence of SEQ ID NO. 2 was used as the first phage, a phage having the genomic DNA sequence of SEQ ID NO. 5 was used as the second phage, and a phage having the genomic DNA sequence of SEQ ID NO. 6 was used as the third phage. Furthermore, the phage possessing the genomic DNA of Sequence ID No. 1 in WO 2024 / 248029 was identified as Xanthomonas campestris pv. vesicatoria (MAFF No. 301256), the phage possessing the genomic DNA of Sequence ID No. 3 was identified as Xanthomonas campestris pv. campestris (MAFF No. 106765), and the other phages were identified using the plaque assay method for Xanthomonas arboricola pv. pruni (MAFF No. 311351) as in Example 1. 8 We confirmed that the level was PFU / mL or higher.

[0179] (3) Evaluation of phage host range The host range of each phage was evaluated using the spot test method. The basic procedure was the same as the method described in "(4) Evaluation of the host range of the first phage" in Example 1. In this example, one type of bacteria was spread on each plate, and the evaluation was performed by dropping the known phage and the first to third phages onto each plate.

[0180] An example of the results is shown in Figures 4 to 10. As shown in Figure 4, consistent with Examples 1 to 3, the first to third phages (labeled "n" to "p" in the figure) showed lytic activity against Xanthomonas campestris pv. vesicatoria (MAFF No. 301256), a species in which some of the known phages (labeled "a" to "m" in the figure) exhibit lytic activity. Furthermore, the results for the known phages (labeled "a" to "m" in the figure) in Figures 4 and 5 were not inconsistent with the lytic activity of each phage shown in WO 2023 / 191074 and WO 2024 / 248029. From this, it was confirmed that this evaluation system is appropriate as an experimental system for simultaneously comparing the lytic activity of each phage. Furthermore, the results in Figure 5 show that the first to third phages (labeled "n" to "p" in the figure) exhibited lytic activity even against Xanthomonas arboricola pv. juglandis, a species against which some known phages (labeled "a" to "m" in the figure) did not show lytic activity. Moreover, it was shown that the phages of the present invention also exhibited lytic activity against bacteria of Xanthomonas campestris pv. vesicatoria and Xanthomonas citri subsp. citri, against which almost all known phages did not show lytic activity (Figures 6 to 10). In particular, it was shown that the phages of the present invention exhibited a characteristic lytic activity that broadly affected bacteria of Xanthomonas citri subsp. citri.

[0181] From the above, it has been shown that the phage of the present invention exhibits lytic activity against a wide range of bacteria compared to known phages, and is particularly effective against various Xanthomonas citri subsp. citri bacteria.

[0182] <Example 5: Further investigation of the lytic activity of the first phage> (Objective) To further investigate the lytic activity of the first phage, which has been confirmed to be effective against bacteria of Xanthomonas citri subsp. citri, including bacterial species of Xanthomonas citri subsp. citri.

[0183] (Methods and Results) In this example, all plant pathogenic bacteria were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in this example are listed in Tables 5 and 6, along with their NARO deposit numbers.

[0184]

[0185]

[0186] The host range of the first phage was evaluated using a spot test method. The basic procedure followed the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0187] Examples of the results are shown in Figures 11 and 12. As shown in Figure 11, all of the first phages were suggested to exhibit lytic activity against a wide range of bacteria of Xanthomonas citri subsp. citri. Furthermore, as shown in Figure 12, the first phages were suggested to exhibit lytic activity not only against Xanthomonas citri subsp. citri, but also against various bacteria of Xanthomonas campestris and Xanthomonas cucurbitae.

[0188] From the above, it was shown that the first phage exhibits lytic activity against a very wide range of bacteria.

[0189] <Example 6: Further investigation of the lytic activity of the second phage> (Objective) To further investigate the lytic activity of the second phage, which has been confirmed to be effective against bacteria of Xanthomonas citri subsp. citri, including bacterial species of Xanthomonas citri subsp. citri.

[0190] (Methods and Results) In this example, all plant pathogenic bacteria were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in this example are listed in Tables 7 and 8, along with their NARO deposit numbers.

[0191]

[0192]

[0193] The host range of the second phage was evaluated using a spot test method. The basic procedure followed the method described in "(4) Evaluation of the host range of the second phage" in Example 1.

[0194] An example of the results is shown in Figures 13 and 14. As shown in Figure 13, the second phage was shown to exhibit lytic activity against a wide range of bacteria in the Xanthomonas citri subsp. citri family. Furthermore, as shown in Figures 13 and 14, it was suggested that the second phage exhibits lytic activity not only against Xanthomonas citri subsp. citri but also against a wide range of bacteria in the Xanthomonas campestris family.

[0195] From the above, it was shown that the second phage exhibits lytic activity against a very wide range of bacteria.

[0196] <Example 7: Further investigation of the lytic activity of the third phage> (Objective) To further investigate the lytic activity of the third phage, which has been confirmed to be effective against bacteria of Xanthomonas citri subsp. citri, including bacterial species of Xanthomonas citri subsp. citri.

[0197] (Methods and Results) In this example, all plant pathogenic bacteria were obtained from the National Agriculture and Food Research Organization (NARO). Table 9 lists each bacterium used in this example along with its NARO deposit numbering.

[0198]

[0199] The host range of the third phage was evaluated using a spot test method. The basic procedure followed the method described in "(4) Evaluation of the host range of the third phage" in Example 1.

[0200] An example of the results is shown in Figure 15. As shown in Figure 15, the third phage not only exhibits lytic activity against a wide range of bacteria including Xanthomonas citri subsp. citri, but is also suggested to exhibit lytic activity against a wide range of bacteria including Xanthomonas campestris pv. campestris.

[0201] From the above, it was shown that the third phage exhibits lytic activity against a wide range of bacteria.

[0202] <Example 8: Investigation of the freezing stability of the phage of the present invention> (Objective) To verify the storage stability of the phage of the present invention.

[0203] (Methods and Results) For the phage solutions of the first to third, each phage purified solution prepared in "(3) Phage Amplification and Purification" of Examples 1 to 3 was diluted to approximately 5.0 × 10⁻¹⁶ in a 50 mM Tris-HCl (pH 7.5) solution. 3 Each dilution was diluted to approximately PFU / mL, and 1.0 mL was dispensed from each dilution for storage at 4°C and for storage at -20°C.

[0204] Each prepared phage solution was dispensed into three vials of 0.2 mL each for storage at 4°C and -20°C. These vials were then stored in a refrigerator at 4°C and a freezer at -20°C for 10 days. After storage, the vials were collected, and the activity of the -20°C stored vials was evaluated after thawing on ice, while the titer of the 4°C stored vials was evaluated by the number of plaques in a plaque assay.

[0205] The plaque assay was basically carried out in accordance with the description in "(2) Isolation and Purification of the First Phage" of Example 1. 50 μL of bacterial culture medium was used as the bacterial suspension, and 10 μL of stored phage solution was used as the phage-containing solution. For the plaque assay, Xanthomonas campestris pv. vesicatoria (MAFF No. 301258) was used for the first phage (having the genomic DNA sequence of SEQ ID NO: 2), and Xanthomonas arboricola pv. juglandis (MAFF No. 212147) was used for the second and third phages.

[0206] The relative residual titer (%) was calculated as the ratio of the titer after storage at -20°C to the titer after storage at 4°C. For each phage, the mean and standard deviation were calculated from nine different relative residual titers, obtained by comparing the titers of three vials stored at -20°C to the titers of three vials stored at 4°C. The titer after storage at 4°C was approximately 5.0 × 10⁻⁶ for all phages used.3 PFU / mL, which was equivalent to the titer before storage.

[0207] An example of the results is shown in Figure 16. As shown in Figure 16, all of Phages 1 to 3 exhibited a titter equal to or higher than that obtained when stored at 4°C even when cryopreserved, and their titer before storage was maintained at a high level, suggesting that they have extremely high cryopreservation stability.

[0208] In general, storage of individual phages at -20°C is susceptible to effects such as the formation of large ice crystals and lacks stability, and it is considered preferable to cryopreserve them at an extremely low temperature of -80°C or lower, or refrigerate them at a temperature of about 4°C (Olson MR, J Virol Methods. 2004;122(2):147-152. doi: 10.1016 / j.jviromet.2004.08.010). However, the phage of the present invention maintained the titer before storage at a level equal to or higher than that obtained after storage at 4°C even after storage at -20°C for 10 days, without being stored in a state where it infected a host or using a cryopreservation solution or the like.

[0209] Since storage at 4°C is generally not considered suitable for extremely long-term storage, special equipment capable of storage at an extremely low temperature is generally required for long-term storage. On the other hand, since the phage of the present invention exhibits extremely high storage stability even when cryopreserved at a relatively high temperature of -20°C, it is considered that it can be stably stored for an extremely long period of time even with simple freezing equipment such as a household freezer.

[0210] From the above, it was suggested that the phage of the present invention is not only characterized by its host range, but also has extremely excellent storage stability, and is unexpectedly useful as a biopesticide. <Example 9: Test for disease control effect of bacterial spot of tomato> (Objective) The effect on plant disease when applied to plants of the isolated novel bacteriophage having lytic activity against pathogenic bacteria of plant disease is verified.

[0211] (Methods) (1) Preparation of phage spray solution The phage spray solution used in this test was prepared according to the following procedure. First, a bacterial culture medium of the host bacterium for phage amplification was prepared according to the following procedure. As the host, Xanthomonas campestris pv. vesicatoria (MAFF No. 301256), a strain in which lytic activity has been confirmed in all phages tested, was used. The bacterial cells of this bacterium were inoculated into YPG Broth and incubated overnight in a shaker set to 25°C. After incubation, OD 600 The turbidity (at a wavelength of 600 nm) was measured, and samples with a turbidity of approximately 1.0 were used as the bacterial culture medium as described below.

[0212] The prepared bacterial culture medium and the phage purification solution containing one type of phage (a novel phage prepared in Examples 1 and 2, and a known phage prepared by the method described in Example 1) (with a titer of 10 8 A mixture of equal volumes of (approximately PFU / mL) and was inoculated into 100 times the volume of YPG culture medium. The culture medium was incubated in a shaker set to 25°C for 8 to 12 hours and collected as the crude phage solution. To the collected crude solution, 1 / 10 the volume of chloroform was added, and after vigorous stirring, the solution was centrifuged at ×8,000 g / 20°C / 5 minutes, and the supernatant was collected. The collected supernatant was passed through a 0.2 μm filter, and the filtrate was used as the phage purification solution.

[0213] As a phage spray solution containing one type of phage, a phage purified solution with a titer of approximately 10 9 A solution diluted with sterile tap water to approximately PFU / mL was used. For phage spray solutions containing multiple types of phages, an equal volume of each phage purified solution, adjusted to have the same order of titer, was mixed, and the titer was approximately 10 9 The solution used was diluted with sterile tap water to a concentration of approximately PFU / mL.

[0214] In this example, in addition to the first phage having the genomic DNA sequence of SEQ ID NO: 2 ("a" in Figure 17) and the second phage having the genomic DNA sequence of SEQ ID NO: 5 ("b" in Figure 17), the following known phages were used: "c" in Figure 17, "d" in Figure 17, "d" in Figure 17, and "e" in Figure 17, "d" in Figure 17,e" in Figure 17, "e" in Figure 17, "e" in Figure 17, "e" in Figure 17, "e" in Figure 17, "e" in Figure 17,

[0215] (2) Preparation of bacterial spray solution The bacterial spray solution used for treating plant infections in this test was prepared using the bacterial culture solution prepared in (1). The bacterial culture solution diluted approximately 10,000 times with sterile tap water was spread onto a YPG Agar plate and incubated in an incubator set to 25°C for 1 to 3 days. The bacterial suspension collected by suspending the colonies on the YPG Agar plate in sterile tap water was used for the final OD 600 The solution was diluted with sterilized tap water to a concentration of approximately 0.5 to prepare the bacterial spray solution.

[0216] (3) Plant specimens: Commercially available tomato seeds were sown and grown in a greenhouse. Seedlings with 50 or more leaves were used as evaluation specimens.

[0217] (4) Phage application and infection treatment In the phage-treated group, each specimen was foliar sprayed with phage solution twice, twice before and twice after bacterial infection treatment, with an interval of 2 to 3 days between each application. Then, 2 to 3 days later, the specimen was foliar sprayed with bacterial solution to infect it with bacteria. The bacterially infected specimens were left to stand in a greenhouse with high humidity for about 2 days after the infection treatment. After that, phage solution was foliar sprayed again twice, with an interval of 2 to 3 days between each application. In the untreated group, the procedure was the same as described above, except that sterile tap water was used instead of phage purified solution.

[0218] (5) Measurement of disease incidence 28 days after infection treatment, when a certain level of disease onset was confirmed in the untreated group, the disease incidence was investigated in the untreated group and each phage-dispersed group.

[0219] Leaves exhibiting the characteristic features of tomato bacterial spot were identified as diseased leaves, and the disease incidence rate was calculated as the ratio of diseased leaves to the total number of leaves. The relative disease incidence rate for the phage-treated group was calculated as a relative value when the disease incidence rate of the untreated group was set to 100%.

[0220] (Results) The results are shown in Figure 17. The disease incidence rate in the untreated group was approximately 32%. On the other hand, the relative disease incidence rate, with the disease incidence rate of the untreated group set at 100%, was approximately 58% when the phage spray solution containing the first phage was applied ("a" in Figure 17), and approximately 52% when the phage spray solution containing the second phage was applied ("b" in Figure 17).

[0221] Furthermore, when a phage spray solution containing two types of phages, the first and second phages, was applied, the relative disease incidence rate decreased even further to an average of approximately 44% (Figure 17, "a / b"). When these phages were combined with three other known phages (a total of five types), the relative disease incidence rate decreased even further to approximately 36% (Figure 17, "a / b / c / d / e"). Generally, the more types of phages combined, the higher the disease control effect and the lower the relative disease incidence rate tended to be.

[0222] From the above, it was found that the phages of the present invention can effectively protect plants from diseases, even when applied to actual plants. Furthermore, it was found that a higher effect can be obtained by applying a combination of the phages of the present invention.

[0223] In all phage application groups, no significant adverse effects on plants that could be attributed to the phage, such as phytotoxicity to the leaves, were observed. This indicates that the application of the phage of the present invention is an effective disease control method with few side effects.

[0224] <Example 10: Disease control efficacy test for broccoli black rot> (Objective) To verify the effect of a newly isolated bacteriophage, which has lytic activity against pathogenic bacteria of plant diseases, on plant diseases when applied to plants.

[0225] (Method) (1) Preparation of phage spray solution The phage spray solution was prepared in accordance with the description in Example 9, except that the bacteria used as the bacterial cells for the phage spray solution was Xanthomonas campestris pv. campestris (MAFF No. 106765) and the following phages were used.

[0226] In this example, a first phage having the genomic DNA sequence of SEQ ID NO: 2 ("a" in Figure 18), a second phage having the genomic DNA sequence of SEQ ID NO: 5 ("b" in Figure 18), and a third phage having the genomic DNA sequence of SEQ ID NO: 6 ("c" in Figure 18) were used in combination with, in addition, a phage having the genomic DNA sequence of SEQ ID NO: 47 in WO 2023 / 191074 (SEQ ID NO: 7) ("d" in Figure 18), a phage having the genomic DNA sequence of SEQ ID NO: 44 in WO 2023 / 191074 (SEQ ID NO: 8) ("e" in Figure 18), a phage having the genomic DNA sequence of SEQ ID NO: 2 in WO 2024 / 248029 (SEQ ID NO: 10) ("f" in Figure 18), and a phage having the genomic DNA sequence of SEQ ID NO: 3 in WO 2024 / 248029 (SEQ ID NO: 11) ("g" in Figure 18). The phage spray solution used included either the first to third phages individually or in combination, or a combination of the first to third phages and four known phages.

[0227] (2) Preparation of bacterial spray solution A phage spray solution was prepared in accordance with the description in Example 9, except that the bacterial cells were the bacteria described above.

[0228] (3) Plant specimens: Commercially available broccoli seeds were sown and grown in a greenhouse. Seedlings with five or more leaves were used as evaluation specimens.

[0229] (4) The application of phages and infection treatment were carried out in accordance with the description in Example 9.

[0230] (5) Measurement of disease incidence The procedure was carried out in accordance with the description in Example 9, except that the evaluation was performed 33 days after infection treatment.

[0231] (Results) The results are shown in Figure 18. The disease incidence rate in the untreated group was approximately 22%. On the other hand, the relative disease incidence rate, with the disease incidence rate of the untreated group set at 100%, was approximately 56% when a phage spray solution containing only one type of phage was applied ("a" in Figure 18), approximately 61% when a phage spray solution containing only one type of phage was applied ("a" in Figure 18), and approximately 50% when a phage spray solution containing only one type of phage was applied ("c" in Figure 18).

[0232] Furthermore, when a phage spray solution containing three or more phages, combining the first to third phages, was applied, the relative disease incidence rate decreased further to an average of approximately 39% (Figure 18, "a / b / c"). In addition to these phages, when four other types of phages (a total of seven types) were combined, the relative disease incidence rate decreased even further to approximately 31% (Figure 18, "a / b / c / d / e / f / g"). Generally, the more types of phages combined, the higher the disease control effect and the lower the relative disease incidence rate tended to be.

[0233] From the above, it was found that even when applied to actual plants, the phages of the present invention can effectively protect plants from diseases, regardless of the type of plant. Furthermore, it was found that a higher effect can be obtained by applying a combination of the phages of the present invention.

[0234] In the phage-treated group, no significant adverse effects on plants that could be attributed to the phage were observed, such as phytotoxicity to the leaves. Therefore, it was found that the application of the phage of the present invention is an effective disease control method with few side effects. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A bacteriophage having a genomic DNA sequence consisting of the base sequence shown in any one of Sequence ID No. 1, 2, or 6.

2. A bacteriophage comprising a bacteriophage whose genomic DNA contains a gene encoding a tail fiber protein having recognition activity for target bacteria, consisting of the amino acid sequence shown in Sequence ID No.

3.

3. The lytic agent according to claim 2, wherein the gene comprises the base sequence shown in Sequence ID No.

4.

4. The lysing agent according to claim 2, wherein the base sequence of the genomic DNA consists of the base sequence shown in Sequence ID No.

5.

5. A lytic agent according to any one of claims 1 to 4, which exhibits lytic activity against bacteria of the genus Xanthomonas.

6. A composition comprising the lysing agent described in claim 5 as an active ingredient.

7. A plant disease control composition comprising the composition described in claim 6.

8. The plant disease control composition according to claim 7, wherein the plant disease is a plant disease caused by bacteria of the genus Xanthomonas.

9. The plant disease control composition according to claim 7, comprising another bacteriophage that exhibits lytic activity against bacteria of the genus Xanthomonas.

10. A method for controlling plant diseases, comprising a contact step of bringing a target plant into contact with the plant disease control composition described in claim 7.

11. A method for identifying bacteria of the genus Xanthomonas, comprising: a culture step of culturing a test bacterium isolated from plant tissue affected by a plant disease to obtain a culture; a mixing step of mixing the culture with the lytic agent described in claim 5 to obtain a mixture; a mixture culture step of culturing the mixture under predetermined conditions; and a determination step of determining that the test bacterium is a bacterium of the genus Xanthomonas if the test bacterium is lysed after the mixture culture step.

12. The method according to claim 11, wherein in the mixture culture step, the mixture further comprises a liquid medium containing soft agar, and the mixture is cultured on a solid medium.

13. The method according to claim 11, wherein in the culture step, the culture comprises a liquid medium containing soft agar, and the culture is cultured on a solid medium.

14. The method according to claim 11, further comprising an isolation step of isolating the test bacteria from plant tissue infected with a plant disease prior to the culture step.

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