Genus xanthomonas bacteriolytic bacteriophage

Abstract

According to the present invention, in order to prevent plant disease caused by bacteria of the genus Xanthomonas, a novel bacteriophage that exhibits bacteriolytic activity specific to bacteria of the genus Xanthomonas is isolated, and a plant disease-preventing composition that uses, as an active ingredient, the novel bacteriophage is developed and provided.　Furthermore, a bacteriolysing agent comprising a bacteriophage that exhibits bacteriolytic activity specific to bacteria of the genus Xanthomonas, the bacteriolysing agent having a novel genomic DNA sequence, and a plant disease-preventing composition comprising the bacteriolysing agent as an active ingredient are provided.

Description

Xanthomonas lytic bacteriophage

[0001] The present invention relates to a lytic agent comprising a bacteriophage, a plant disease control composition containing the same, and a plant disease control method.

[0002] Bacteriophages (often abbreviated as "phages" herein) are a general term for viruses that infect only bacteria. Many phages, also known as lytic phages, specifically adsorb to their target host bacteria, inject their own DNA, and self-amplify using the bacterial translation machinery. They then lyse the bacteria, spreading the amplified phages and repeatedly infecting new target bacteria (Non-Patent Document 1).

[0003] Many Xanthomonas bacteria are known to cause diseases of various crops and other plants, and copper compounds and antibiotics have been used as common control methods. However, due to many problems, such as inefficacy, phytotoxicity, and the potential for disruption of the bacterial flora balance, phage-based methods have recently attracted attention as a new control method (Non-Patent Document 2).

[0004] Phages that are lytic to Xanthomonas bacteria have been reported, for example, in Patent Documents 1 to 3 and Non-Patent Document 2. However, because the host range of phages is extremely narrow, it remains important to search for new phages and discover phages with higher lytic activity.

[0005] JP 2016-32435 A JP 2021-102635 A 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 are viruses and natural products, and no reports of phytotoxicity have been reported to date. Furthermore, because phages have extremely high host specificity, they target only specific genera and species of bacteria, and their impact on the balance of the microflora is extremely limited. Furthermore, they are harmless to not only humans and other animals, but also plants, making them highly safe.

[0008] Therefore, in order to prevent damage to agricultural crops caused by plant diseases caused by Xanthomonas bacteria, we have isolated a novel phage that exhibits lytic activity against Xanthomonas bacteria, and we also aim to provide a composition containing the phage as an active ingredient, which can be used for disease control, pathogenic bacteria detection, etc.

[0009] The present inventors isolated novel phages from natural wastewater and soil using a technique for detecting lytic plaques formed on soft agar medium cultured with Xanthomonas bacteria, evaluated the lytic activity of the phages against various Xanthomonas bacteria, and analyzed their genome sequences. As a result, it was revealed that the phages possess novel genome DNA sequences. The present invention was completed based on the results of the above research and development, and specifically provides the following:

[0010] (1-1) A lytic agent comprising a bacteriophage having in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the lytic agent comprising any one of the amino acid sequences shown in any one of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 1; (b) an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. (1-2) The lytic agent according to (1-1), wherein the amino acid sequence shown in SEQ ID NO: 1 is any one of the amino acid sequences shown in SEQ ID NOs: 2 to 4. (1-3) A lytic agent according to (1-1) or (1-2), wherein the gene consists of any one of the following base sequences (d) to (f): (d) a base sequence shown in any one of SEQ ID NOs: 5 to 7; (e) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in any one of SEQ ID NOs: 5 to 7; (f) a base sequence having 90% or more sequence identity with the base sequence shown in any one of SEQ ID NOs: 5 to 7. (1-4) A lytic agent according to any one of (1-1) to (1-3), wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (g) to (k) below: (g) a base sequence shown in any one of SEQ ID NOs: 8 to 10; (h) a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the gene base sequence shown in any one of (1-1) to (1-3) in the base sequence shown in any one of SEQ ID NOs: 8 to 10; (i) a base sequence in which a base sequence other than the gene base sequence shown in any one of (1-1) to (1-3) in the base sequence shown in any one of SEQ ID NOs: 8 to 10 has 80% or more sequence identity; (j) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in any one of SEQ ID NOs: 8 to 10; (k) a base sequence in which a base sequence in which one or more bases have been added, deleted, and / or substituted in any one of SEQ ID NOs: 8 to 10 has 90% or more sequence identity. (1-5) The bacteriolytic agent according to any one of (1-1) to (1-4), which exhibits bacteriolytic activity against Xanthomonas bacteria.(1-6) The bacteriolytic agent according to (1-5), wherein the Xanthomonas bacterium is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, Xanthomonas citri, Xanthomonas oryzae, and Xanthomonas cucurbitae.

[0011] (2-1) A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the gene comprising the amino acid sequence shown in either (a) or (b) below: (a) the amino acid sequence shown in SEQ ID NO: 11 or 45, or (b) the amino acid sequence shown in SEQ ID NO: 11 in which one amino acid other than that at positions 278 and 350 has been substituted. (2-2) The lytic agent according to (2-1), wherein the gene comprises the base sequence shown in SEQ ID NO: 12 or 46. (2-3) A lytic agent according to (2-1) or (2-2), wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (a) to (e) below: (a) the base sequence shown in SEQ ID NO: 13, 47 or 48; (b) a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the gene base sequence shown in (2-1) or (2-2) in the base sequence shown in SEQ ID NO: 13, 47 or 48; (c) a base sequence in which the base sequence shown in SEQ ID NO: 13, 47 or 48 has a sequence identity of 98.5% or more with a base sequence other than the gene base sequence shown in (2-1) or (2-2) in the base sequence shown in SEQ ID NO: 13, 47 or 48; (d) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 13, 47 or 48; (e) a base sequence in which the base sequence shown in SEQ ID NO: 13, 47 or 48 has a sequence identity of 98.5% or more. (2-4) The bacteriolytic agent according to any one of (2-1) to (2-3), which exhibits bacteriolytic activity against Xanthomonas bacteria. (2-5) The bacteriolytic agent according to (2-4), wherein the Xanthomonas bacteria is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, Xanthomonas citri, Xanthomonas oryzae, and Xanthomonas cucurbitae.

[0012] (3-1) A lytic agent comprising a bacteriophage having a genomic DNA sequence containing any one of the following nucleotide sequences (a) to (c): (a) the nucleotide sequence shown in SEQ ID NO: 14; (b) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 14; (c) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 14. (3-2) The lytic agent according to (3-1), which exhibits lytic activity against bacteria of the genus Xanthomonas. (3-3) The lytic agent according to (3-2), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0013] (4-1) A lytic agent comprising a bacteriophage having in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the gene comprising the amino acid sequence shown in any one of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 15; (b) the amino acid sequence shown in SEQ ID NO: 15 with one or more amino acids added, deleted, and / or substituted; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 15. (4-2) The lytic agent according to (4-1), wherein the gene comprises any one of the nucleotide sequences shown in (d) to (f) below: (d) the nucleotide sequence shown in SEQ ID NO: 16; (e) the nucleotide sequence shown in SEQ ID NO: 16 with one or more nucleotides added, deleted, and / or substituted; (f) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 16. (4-3) The bacteriolytic agent according to (4-1) or (4-2), wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (g) to (k) below: (g) the base sequence shown in SEQ ID NO: 17; (h) the base sequence shown in SEQ ID NO: 17, in which one or more bases have been added, deleted, and / or substituted to a base sequence other than the gene base sequence shown in (4-1) or (4-2); (i) the base sequence shown in SEQ ID NO: 17, in which a base sequence other than the gene base sequence shown in (4-1) or (4-2) has 80% or more sequence identity; (j) the base sequence shown in SEQ ID NO: 17, in which one or more bases have been added, deleted, and / or substituted; (k) a base sequence that has 90% or more sequence identity to the base sequence shown in SEQ ID NO: 17. (4-4) The bacteriolytic agent according to any one of (4-1) to (4-3), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas. (4-5) The lytic agent according to (4-4), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0014] (5-1) A lytic agent comprising a bacteriophage having a genomic DNA sequence containing any one of the following nucleotide sequences (a) to (c): (a) the nucleotide sequence shown in SEQ ID NO: 18 or 19; (b) the nucleotide sequence shown in SEQ ID NO: 18 or 19 in which one or more nucleotides have been added, deleted, and / or substituted; or (c) a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 18 or 19. (5-2) The lytic agent according to (5-1), which exhibits lytic activity against bacteria of the genus Xanthomonas. (5-3) The lytic agent according to (5-2), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0015] (6-1) A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a protein consisting of any one of the amino acid sequences shown in (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 21; (b) the amino acid sequence shown in SEQ ID NO: 21 with one or more amino acids added, deleted, and / or substituted; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21. (6-2) The lytic agent according to (6-1), wherein the gene consists of any one of the nucleotide sequences shown in (d) to (f) below: (d) the nucleotide sequence shown in SEQ ID NO: 22; (e) the nucleotide sequence shown in SEQ ID NO: 22 with one or more nucleotides added, deleted, and / or substituted; (f) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 22. (6-3) The bacteriolytic agent according to (6-1) or (6-2), wherein the base sequence of the genomic DNA consists of any one of the base sequences (g) to (k) below: (g) the base sequence shown in SEQ ID NO: 23; (h) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 23 other than the gene described in (6-1) or (6-2) above; (i) a base sequence in which the base sequence shown in SEQ ID NO: 23 has 95% or more sequence identity with a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 23 other than the gene described in (6-1) or (6-2) above: (j) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 23; (k) a base sequence in which the base sequence has 95% or more sequence identity with the base sequence shown in SEQ ID NO: 23. (6-4) The bacteriolytic agent according to any one of (6-1) to (6-3), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas. (6-5) The lytic agent according to (6-4), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0016] (7-1) A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a tail tube protein A having the activity of recognizing a target bacterium, the gene having the amino acid sequence shown in any one of (a) to (c) below, and a gene encoding a tail tube protein B having the activity of recognizing a target bacterium, the gene having the amino acid sequence shown in any one of (d) to (f) below: (a) the amino acid sequence shown in SEQ ID NO: 24, 49, or 60; (b) the amino acid sequence shown in SEQ ID NO: 24 or 49 in which one or more amino acids have been added, deleted, and / or substituted; (c) an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24 or 49; (d) the amino acid sequence shown in SEQ ID NO: 57, 50, or 61; (e) the amino acid sequence shown in SEQ ID NO: 57 or 50 in which one or more amino acids have been added, deleted, and / or substituted; (f) an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 57 or 50. (7-2) The lytic agent according to (7-1), wherein the gene encoding tail tube protein A consists of any one of the following nucleotide sequences (g) to (i): (g) the nucleotide sequence shown in SEQ ID NO: 26 or 51; (h) the nucleotide sequence shown in SEQ ID NO: 26 or 51, in which one or more nucleotides have been added, deleted, and / or substituted; (i) a nucleotide sequence having 97% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 26 or 51. (7-3) The lytic agent according to (7-1) or (7-2), wherein the gene encoding tail tube protein B consists of any one of the following nucleotide sequences (j) to (l): (j) the nucleotide sequence shown in SEQ ID NO: 27 or 52; (k) the nucleotide sequence shown in SEQ ID NO: 27 or 52, in which one or more nucleotides have been added, deleted, and / or substituted; (l) a nucleotide sequence having 97% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 27 or 52.(7-4) A lytic agent according to any one of (7-1) to (7-3), wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (m) to (q) below: (m) the base sequence shown in SEQ ID NO: 28, 29 or 53; (n) a base sequence in which one or several bases have been added, deleted, and / or substituted in a base sequence other than the gene base sequence shown in any one of (7-1) to (7-3) in the base sequence shown in SEQ ID NO: 28, 29 or 53; (o) a base sequence in which the base sequence shown in SEQ ID NO: 28, 29 or 53 has 95% or more sequence identity with a base sequence other than the gene set forth in any one of (7-1) to (7-3): (p) a base sequence in which one or several bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 28, 29 or 53; (q) a base sequence in which the base sequence shown in SEQ ID NO: 28, 29 or 53 has 95% or more sequence identity. (7-5) The bacteriolytic agent according to any one of (7-1) to (7-4), which exhibits bacteriolytic activity against Xanthomonas bacteria. (7-6) The bacteriolytic agent according to (7-5), wherein the Xanthomonas bacteria is Xanthomonas arboricola or Xanthomonas campestris.

[0017] (8-1) A lytic agent comprising a bacteriophage having a genomic DNA sequence containing any one of the following nucleotide sequences (a) to (c): (a) the nucleotide sequence shown in any one of SEQ ID NOs: 32 to 36; (b) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in any one of SEQ ID NOs: 32 to 36; (c) a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in any one of SEQ ID NOs: 32 to 36. (8-2) The lytic agent according to (8-1), which exhibits lytic activity against bacteria of the genus Xanthomonas. (8-3) The lytic agent according to (8-2), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0018] (9-1) A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a tail tube protein A having the activity of recognizing a target bacterium, the gene having the amino acid sequence shown in any one of (a) to (c) below, and a gene encoding a tail tube protein B having the activity of recognizing a target bacterium, the gene having the amino acid sequence shown in any one of (d) to (f) below: (a) the amino acid sequence shown in SEQ ID NO: 37; (b) the amino acid sequence shown in SEQ ID NO: 37 in which one or more amino acids have been added, deleted, and / or substituted; (c) an amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 37; (d) the amino acid sequence shown in SEQ ID NO: 38, 54, or 59; (e) the amino acid sequence shown in SEQ ID NO: 38 or 54 in which one or more amino acids have been added, deleted, and / or substituted; (f) an amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 38 or 54. (9-2) The lytic agent according to (9-1), wherein the gene encoding tail tube protein A consists of any one of the following nucleotide sequences (g) to (i): (g) the nucleotide sequence shown in SEQ ID NO: 39; (h) the nucleotide sequence shown in SEQ ID NO: 39 with one or more bases added, deleted, and / or substituted; (i) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 39. (9-3) The lytic agent according to (9-1) or (9-2), wherein the gene encoding tail tube protein B consists of any one of the following nucleotide sequences (j) to (l): (j) the nucleotide sequence shown in SEQ ID NO: 40 or 55; (k) the nucleotide sequence shown in SEQ ID NO: 40 or 55 with one or more bases added, deleted, and / or substituted; (l) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 40 or 55.(9-4) A lytic agent according to any one of (9-1) to (9-3), wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (m) to (q) below: (m) the base sequence shown in SEQ ID NO: 41 or 56; (n) a base sequence in which one or several bases have been added, deleted, and / or substituted in a base sequence other than the gene base sequence shown in any one of (9-1) to (9-3) in the base sequence shown in SEQ ID NO: 41 or 56; (o) a base sequence in which the base sequence shown in SEQ ID NO: 41 or 56 has 80% or more sequence identity with a base sequence other than the gene set forth in any one of (9-1) to (9-3): (p) a base sequence in which one or several bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 41 or 56; (q) a base sequence in which the base sequence shown in SEQ ID NO: 41 or 56 has 90% or more sequence identity. (9-5) The bacteriolytic agent according to any one of (9-1) to (9-4), which exhibits bacteriolytic activity against Xanthomonas bacteria. (9-6) The bacteriolytic agent according to (9-5), wherein the Xanthomonas bacteria is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, and Xanthomonas citri.

[0019] (10-1) A lytic agent comprising a bacteriophage having in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the gene comprising the amino acid sequence shown in any one of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 42; (b) the amino acid sequence shown in SEQ ID NO: 42 in which one or more amino acids have been added, deleted, and / or substituted; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 42. (10-2) The lytic agent according to (10-1), wherein the gene comprises any one of the nucleotide sequences shown in SEQ ID NO: 43 below: (d) the nucleotide sequence shown in SEQ ID NO: 43; (e) the nucleotide sequence shown in SEQ ID NO: 43 in which one or more nucleotides have been added, deleted, and / or substituted; (f) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 43. (10-3) A lytic agent according to (10-1) or (10-2), wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (g) to (k) below: (g) the base sequence shown in SEQ ID NO: 44; (h) a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the gene base sequence shown in (10-1) or (10-2) in the base sequence shown in SEQ ID NO: 44; (i) a base sequence in which the base sequence shown in SEQ ID NO: 44 has 80% or more sequence identity with a base sequence other than the gene base sequence shown in (10-1) or (10-2); (j) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 44; (k) a base sequence in which the base sequence shown in SEQ ID NO: 44 has 90% or more sequence identity. (10-4) The bacteriolytic agent according to any one of (10-1) to (10-3), which exhibits bacteriolytic activity against Xanthomonas bacteria. (10-5) The bacteriolytic agent according to (10-4), wherein the Xanthomonas bacteria is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, and Xanthomonas citri.

[0020] [1-1] A lytic agent comprising a bacteriophage having in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the bacteriophage having an amino acid sequence shown in any one of the following (a) to (d): (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 45, 42, and 1; (b) an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 42 or 1; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 42 or 1; or (d) an amino acid sequence in which one amino acid other than at positions 278 and 350 in the amino acid sequence shown in SEQ ID NO: 11 has been substituted. [1-2] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene consisting of an amino acid sequence shown in any one of (e) to (g) below, and a gene consisting of an amino acid sequence shown in any one of (h) to (j) below, wherein the genes are a gene encoding a tailtube protein A and a gene encoding a tailtube protein B, each of which has the activity of recognizing a target bacterium, said lytic agent being: (e) the amino acid sequence shown in SEQ ID NO: 24, 49, or 60; (f) the amino acid sequence shown in SEQ ID NO: 24 or 49 in which one or more amino acids have been added, deleted, and / or substituted; (g) an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24 or 49; (h) the amino acid sequence shown in SEQ ID NO: 57, 50, or 61; (i) the amino acid sequence shown in SEQ ID NO: 57 or 50 in which one or more amino acids have been added, deleted, and / or substituted; or (j) an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 57 or 50.[1-3] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene consisting of any one of the amino acid sequences (k) to (m) below and a gene consisting of any one of the amino acid sequences (n) to (p) below, wherein the genes are a gene encoding a tailtube protein A and a gene encoding a tailtube protein B, each of which has the activity of recognizing a target bacterium, said lytic agent being: (k) the amino acid sequence shown in SEQ ID NO: 37; (l) the amino acid sequence shown in SEQ ID NO: 37 with one or more amino acids added, deleted, and / or substituted; (m) an amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 37; (n) the amino acid sequence shown in SEQ ID NO: 38, 54, or 59; (o) the amino acid sequence shown in SEQ ID NO: 38 or 54 with one or more amino acids added, deleted, and / or substituted; or (p) an amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 38 or 54. [1-4] The bacteriolytic agent according to [1-1], wherein the amino acid sequence shown in SEQ ID NO: 1 is any one of the amino acid sequences shown in SEQ ID NOs: 2 to 4. [1-5] The bacteriolytic agent according to [1-1], wherein the gene consists of any one of the following nucleotide sequences (1) to (3): (1) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12, 46, 43, and 5 to 7, (2) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence selected from the group consisting of SEQ ID NOs: 43 and 5 to 7, or (3) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence selected from the group consisting of SEQ ID NOs: 43 and 5 to 7.[1-6] The lytic agent according to [1-5], wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (1') to (7') below: (1') a base sequence selected from the group consisting of SEQ ID NOs: 13, 47, 48, 44, and 8 to 10; (2') a base sequence selected from the group consisting of SEQ ID NOs: 13, 47, 48, 44, and 8 to 10, in which one or more bases have been added, deleted, and / or substituted in the base sequence other than the gene according to [1-5]; (3') a base sequence selected from the group consisting of SEQ ID NOs: 44 and 8 to 10, in which 80% of the base sequence other than the gene according to [1-5] (4') a base sequence having 98.5% or more sequence identity to the base sequence shown in SEQ ID NO: 13, 47 or 48 of a base sequence other than the gene described in [1-5]; (5') a base sequence having one or more bases added, deleted, and / or substituted in the base sequence shown in any one of SEQ ID NOs: 13, 47, 48, 44 and 8 to 10; or (6') a base sequence having 90% or more sequence identity to the base sequence shown in any one of SEQ ID NOs: 44 and 8 to 10; (7') a base sequence having 98.5% or more sequence identity to the base sequence shown in SEQ ID NO: 13, 47 or 48. [1-7] The lytic agent according to [1-2], wherein the gene encoding tail tube protein A consists of any one of the following nucleotide sequences (4) to (6): (4) the nucleotide sequence shown in SEQ ID NO: 26 or 51, (5) the nucleotide sequence shown in SEQ ID NO: 26 or 51 in which one or more nucleotides have been added, deleted, and / or substituted, or (6) a nucleotide sequence having 97% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 26 or 51. [1-8] The lytic agent according to [1-2] or [1-7], wherein the gene encoding tail tube protein B consists of any one of the following nucleotide sequences (7) to (9): (7) the nucleotide sequence shown in SEQ ID NO: 27 or 52, (8) the nucleotide sequence shown in SEQ ID NO: 27 or 52 in which one or more nucleotides have been added, deleted, and / or substituted, or (9) a nucleotide sequence having 97% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 27 or 52.[1-9] The lytic agent according to [1-2], wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (8') to (12') below: (8') the base sequence shown in SEQ ID NO: 28, 29 or 53, (9') the base sequence shown in SEQ ID NO: 28, 29 or 53 in which one or more bases have been added, deleted, and / or substituted to a base sequence other than the gene described in [1-2], (10') the base sequence shown in SEQ ID NO: 28, 29 or 53, which has 95% or more sequence identity to a base sequence other than the gene described in [1-2], (11') the base sequence shown in SEQ ID NO: 28, 29 or 53 in which one or more bases have been added, deleted, and / or substituted, or (12') the base sequence which has 95% or more sequence identity to a base sequence shown in SEQ ID NO: 28, 29 or 53. [1-10] The lytic agent according to [1-3], wherein the gene encoding tail tube protein A consists of any one of the following nucleotide sequences (10) to (12): (10) the nucleotide sequence shown in SEQ ID NO: 39, (11) the nucleotide sequence shown in SEQ ID NO: 39 in which one or more nucleotides have been added, deleted, and / or substituted, or (12) a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 39. [1-11] The lytic agent according to [1-3] or [1-10], wherein the gene encoding tail tube protein B consists of any one of the following nucleotide sequences (13) to (15): (13) the nucleotide sequence shown in SEQ ID NO: 40 or 55, (14) the nucleotide sequence shown in SEQ ID NO: 40 or 55 in which one or more nucleotides have been added, deleted, and / or substituted, or (15) a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 40 or 55.[1-12] The lytic agent according to [1-3], wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (13') to (17') below: (13') the base sequence shown in SEQ ID NO: 41 or 56, (14') the base sequence shown in SEQ ID NO: 41 or 56 in which one or more bases have been added, deleted, and / or substituted to a base sequence other than the gene described in [1-3], (15') the base sequence shown in SEQ ID NO: 41 or 56, which has 80% or more sequence identity to a base sequence other than the gene described in [1-3], (16') the base sequence shown in SEQ ID NO: 41 or 56 in which one or more bases have been added, deleted, and / or substituted, or (17') the base sequence which has 90% or more sequence identity to the base sequence shown in SEQ ID NO: 41 or 56. [1-13] The bacteriolytic agent according to any one of [1-1] to [1-12], which exhibits bacteriolytic activity against Xanthomonas bacteria.

[0021] [2-1] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene consisting of any one of the amino acid sequences (a) to (c) below and a gene consisting of any one of the amino acid sequences (d) to (f) below, wherein the genes are a gene encoding a tail tube protein A and a gene encoding a tail tube protein B, each of which has the activity of recognizing a target bacterium, said lytic agent being: (a) the amino acid sequence shown in SEQ ID NO: 24, 49 or 60; (b) the amino acid sequence shown in SEQ ID NO: 24 or 49 in which one or more amino acids have been added, deleted, and / or substituted; (c) an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 24 or 49; (d) the amino acid sequence shown in SEQ ID NO: 57, 50 or 61; (e) the amino acid sequence shown in SEQ ID NO: 57 or 50 in which one or more amino acids have been added, deleted, and / or substituted; or (f) an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 57 or 50. [2-2] A lytic agent comprising a bacteriophage having in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the bacteriophage having any one of the amino acid sequences shown in any one of (g) to (j) below: (g) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 11, 45, 15, and 42; (h) an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 11, 15, and 42; (i) an amino acid sequence having 90% or more sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 11, 15, and 42; or (j) an amino acid sequence in which one amino acid other than at positions 278 and 350 in the amino acid sequence shown in SEQ ID NO: 11 has been substituted.[2-3] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene consisting of any one of the amino acid sequences (k) to (m) below and a gene consisting of any one of the amino acid sequences (n) to (p) below, wherein the genes are a gene encoding a tailtube protein A and a gene encoding a tailtube protein B, each of which has the activity of recognizing a target bacterium, the lytic agent being: (k) the amino acid sequence shown in SEQ ID NO: 37; (l) the amino acid sequence shown in SEQ ID NO: 37 with one or more amino acids added, deleted, and / or substituted; (m) an amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 37; (n) the amino acid sequence shown in SEQ ID NO: 38, 54, or 59; (o) the amino acid sequence shown in SEQ ID NO: 38 or 54 with one or more amino acids added, deleted, and / or substituted; or (p) an amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 38 or 54. [2-4] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a protein consisting of any one of the following amino acid sequences (q) to (s): (q) the amino acid sequence shown in SEQ ID NO: 21, (r) the amino acid sequence shown in SEQ ID NO: 21 in which one or more amino acids have been added, deleted, and / or substituted, or (s) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21. [2-5] A lytic agent comprising a bacteriophage having a genomic DNA sequence containing any one of the following base sequences (1') to (3'): (1') a base sequence selected from the group consisting of SEQ ID NOs: 14, 18, 19, and 32 to 36, (2') a base sequence in which one or more bases have been added, deleted, and / or substituted in any one of the base sequences selected from the group consisting of SEQ ID NOs: 14, 18, 19, and 32 to 36, or (3') a base sequence having 90% or more sequence identity with any one of the base sequences selected from the group consisting of SEQ ID NOs: 14, 18, 19, and 32 to 36.[2-6] The lytic agent according to [2-1], wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (4') to (8') below: (4') the base sequence shown in SEQ ID NO: 28, 29 or 53; (5') the base sequence shown in SEQ ID NO: 28, 29 or 53, in which one or more bases have been added, deleted, and / or substituted to a base sequence other than the gene described in [2-2]; (6') the base sequence shown in SEQ ID NO: 28, 29 or 53, which has 95% or more sequence identity to a base sequence other than the gene described in [2-2]; (7') the base sequence shown in SEQ ID NO: 28, 29 or 53, in which one or more bases have been added, deleted, and / or substituted; or (8') the base sequence shown in SEQ ID NO: 28, 29 or 53, which has 95% or more sequence identity. [2-7] The bacteriolytic agent according to any one of [2-1] to [2-6], which exhibits bacteriolytic activity against Xanthomonas bacteria.

[0022] [3-1] A lytic agent comprising a bacteriophage having a genomic DNA sequence containing any one of the following nucleotide sequences (1') to (3'): (1') a nucleotide sequence selected from the group consisting of SEQ ID NOs: 32 to 36, 14, 18, and 19, (2') a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 32 to 36, 14, 18, and 19, or (3') a nucleotide sequence having 90% or more sequence identity with any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 32 to 36, 14, 18, and 19. [3-2] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the tail fiber protein consisting of any one of the amino acid sequences shown in (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 15, (b) the amino acid sequence shown in SEQ ID NO: 15 with one or more amino acids added, deleted, and / or substituted, or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 15. [3-3] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a protein consisting of any one of the amino acid sequences shown in (d) to (f) below: (d) the amino acid sequence shown in SEQ ID NO: 21, (e) the amino acid sequence shown in SEQ ID NO: 21 with one or more amino acids added, deleted, and / or substituted, or (f) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 21. [3-4] The lytic agent according to [3-2], wherein the gene consists of a base sequence shown in any one of the following (1) to (3): (1) the base sequence shown in SEQ ID NO: 16, (2) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 16, or (3) a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 16.[3-5] The lytic agent according to [3-2], wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (4') to (8') below: (4') the base sequence shown in SEQ ID NO: 17, (5') the base sequence shown in SEQ ID NO: 17 in which one or more bases have been added, deleted, and / or substituted to a base sequence other than the gene described in [3-2], (6') the base sequence shown in SEQ ID NO: 17 in which the base sequence other than the gene described in [3-2] has 80% or more sequence identity, (7') the base sequence shown in SEQ ID NO: 17 in which one or more bases have been added, deleted, and / or substituted, or (8') the base sequence in which the base sequence shown in SEQ ID NO: 17 has 90% or more sequence identity. [3-6] The lytic agent according to [3-3], wherein the gene consists of any of the following base sequences (4) to (6): (4) the base sequence shown in SEQ ID NO: 22, (5) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 22, or (6) a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 22. [3-7] The bacteriolytic agent according to [3-3], wherein the base sequence of the genomic DNA consists of any one of the base sequences (9') to (13') below: (9') the base sequence shown in SEQ ID NO: 23, (10') the base sequence shown in SEQ ID NO: 23 in which one or more bases have been added, deleted, and / or substituted to a base sequence other than the gene described in [3-3], (11') the base sequence shown in SEQ ID NO: 23, which has 95% or more sequence identity to a base sequence other than the gene described in [3-3], (12') the base sequence shown in SEQ ID NO: 23 in which one or more bases have been added, deleted, and / or substituted, or (13') the base sequence which has 95% or more sequence identity to the base sequence shown in SEQ ID NO: 23. [3-8] The bacteriolytic agent according to any of [3-1] to [3-7], which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas. [3-9] The bacteriolytic agent according to [3-8], wherein the Xanthomonas bacterium is Xanthomonas arboricola. [3-10] The bacteriolytic agent according to [3-9], wherein the pathotype of the Xanthomonas bacterium is pruni.

[0023] [4-1] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the tail fiber protein consisting of any one of the amino acid sequences shown in SEQ ID NOs: 42, 11, 45, and 15. [4-2] The lytic agent according to [4-1], wherein the gene consists of any one of the base sequences shown in SEQ ID NOs: 43, 12, 46, and 16. [4-3] The lytic agent according to [4-1], wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in SEQ ID NOs: 44, 13, 47, 48, and 17. [4-4] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, the tail fiber protein consisting of the amino acid sequence shown in SEQ ID NO: 1. [4-5] The lytic agent according to [4-4], wherein the amino acid sequence shown in SEQ ID NO: 1 is any one of the amino acid sequences shown in SEQ ID NOs: 2 to 4. [4-6] The bacteriolytic agent according to [4-4], wherein the gene consists of a nucleotide sequence shown in any one of SEQ ID NOs: 5 to 7. [4-7] The bacteriolytic agent according to [4-4], wherein the nucleotide sequence of the genomic DNA consists of a nucleotide sequence shown in any one of SEQ ID NOs: 8 to 10. [4-8] A bacteriolytic agent comprising a bacteriophage having a genomic DNA sequence including a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 18, 19, and 32 to 36. [4-9] A bacteriolytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 21. [4-10] The bacteriolytic agent according to [4-9], wherein the gene consists of a nucleotide sequence shown in SEQ ID NO: 22. [4-11] The bacteriolytic agent according to [4-9], wherein the nucleotide sequence of the genomic DNA consists of the nucleotide sequence shown in SEQ ID NO: 23. [4-12] A lytic agent comprising a bacteriophage containing in its genomic DNA a gene encoding tailtube protein A, which has the amino acid sequence shown in SEQ ID NO: 24, 49, or 37 and has the activity of recognizing a target bacterium, and a gene encoding tailtube protein B, which has the amino acid sequence shown in any one of the group consisting of SEQ ID NOs: 57, 50, 38, 54, and 59 and has the activity of recognizing a target bacterium.[4-13] The bacteriolytic agent according to [4-12], wherein the gene encoding tailtube protein A consists of the nucleotide sequence shown in SEQ ID NO: 26, 51, or 39. [4-14] The bacteriolytic agent according to [4-12], wherein the gene encoding tailtube protein B consists of the nucleotide sequence shown in any one of SEQ ID NOs: 27, 52, 40, and 55. [4-15] The bacteriolytic agent according to [4-12], wherein the nucleotide sequence of the genomic DNA consists of the nucleotide sequence shown in any one of SEQ ID NOs: 28, 29, 53, or 41. [4-16] The bacteriolytic agent according to any one of [4-1] to [4-15], which exhibits lytic activity against bacteria of the genus Xanthomonas.

[0024] <1> A composition comprising one or more lytic agents according to any one of (1-1) to (10-5) and [1-1] to [4-16] as an active ingredient. <2> A composition comprising two or more lytic agents according to any one of (1-1) to (10-5) and [1-1] to [4-16] as active ingredients. <3> A plant disease control composition comprising the composition according to <1> or <2>. <4> The plant disease control composition according to <3>, which is for a plant disease caused by bacteria of the genus Xanthomonas. <5> The plant disease control composition according to <3> or <4>, which contains another bacteriophage that exhibits lytic activity against bacteria of the genus Xanthomonas. <6> A plant disease control method comprising a contacting step of contacting a target plant with the plant disease control composition according to any one of <3> to <5>. <7> A method for identifying Xanthomonas bacteria, comprising: a culturing step of culturing a test bacterium isolated from plant tissue infected with a plant disease to obtain a culture; a mixing step of mixing the culture with a lysing agent described in any one of (1-1) to (10-5) and [1-1] to [4-16] to obtain a mixture; a mixture culturing step of culturing the mixture under predetermined conditions; and a determination step of determining that the test bacterium is a Xanthomonas bacterium if the test bacterium is lysed after the mixture culturing step. <8> The method described in <7>, wherein in the mixture culturing step, the mixture further comprises a soft agar-containing liquid medium, and the mixture is cultured on a solid medium. <9> The method described in <7>, wherein in the culturing step, the culture comprises a soft agar-containing liquid medium, and the culture is cultured on a solid medium. <10> The method described in any one of <7> to <9>, further comprising an isolation step of isolating the test bacterium from plant tissue infected with a plant disease before the culturing step. This specification includes the disclosures of Japanese Patent Application Nos. 2022-059936, 2022-060143, 2022-060249, 2022-060441, 2022-060819, 2022-060822, 2022-060887, 2022-060909, 2022-061075, 2022-156882, 2022-156972, 2022-157086, 2023-041699, 2023-041776, 2023-041784, and 2023-041827, which are priority documents of the present application.

[0025] The bacteriolytic agent of the present invention and a composition containing it as an active ingredient can lyse specific target bacteria.

[0026] The plant disease control composition of the present invention can prevent and suppress diseases caused by specific target bacteria.

[0027] 1A and 1B show the lytic activity of the first bacteriophage obtained in Example 1. (A) is a diagram showing agar plates in which Xanthomonas bacteria shown in Table 2 and control Pseudomonas fluorescens were cultured, followed by dropping the first phage purified solution onto the plates and statically culturing. (B) is a diagram of the plates corresponding to (A), showing the position of the phage purified solution dropped onto each plate. In (A) and (B), (1) indicates a plate on which bacteria with an ID of MAFF No. 211191 were spread; (2) indicates a plate on which bacteria with an ID of MAFF No. 311351 were spread; (3) indicates a plate on which bacteria with an ID of MAFF No. 301256 were spread; (4) indicates a plate on which bacteria with an ID of MAFF No. 106765 were spread; (5) indicates a plate on which bacteria with an ID of MAFF No. 301078 were spread; and (6) indicates a plate on which control Pseudomonas fluorescens with an ID of NCIMB-ID 10460 were spread. 1B, "a" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 8, "b" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 9, and "c" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 10. This figure shows the lytic activity of the second bacteriophage obtained in Example 2. (A) is a diagram showing Xanthomonas bacteria and control Pseudomonas fluorescens cultured on an agar plate, after which the second phage purified solution was dropped onto the center of the plate and allowed to stand. (B) is a diagram showing the plates corresponding to (A), showing the bacteria shown in Table 4 spread on each plate. This figure shows the lytic activity of the third bacteriophage obtained in Example 3. (A) is a diagram showing Xanthomonas bacteria and control Pseudomonas fluorescens cultured on an agar plate, after which the third phage purified solution was dropped onto the plate and allowed to stand. B is a plate diagram corresponding to A, showing the positions of the purified phage solution dropped onto each plate.3A and 3B, 1 indicates a plate on which bacteria with an ID of MAFF No. 311282 were spread, 2 indicates a plate on which bacteria with an ID of MAFF No. 301426 were spread, 3 indicates a plate on which bacteria with an ID of MAFF No. 311351 were spread, 4 indicates a plate on which bacteria with an ID of MAFF No. 311586 were spread, 5 indicates a plate on which bacteria with an ID of MAFF No. 311618 were spread, and 6 indicates a plate on which control Pseudomonas fluorescens with an ID of NCIMB-ID 10460 was spread. These figures show the lytic activity of the fourth bacteriophage obtained in Example 4. Figure A shows a diagram of Xanthomonas bacteria and control Pseudomonas fluorescens spread on an agar plate, followed by the addition of a drop of the fourth phage purified solution to the center of the plate and static culture. Figure B is a diagram of the plate corresponding to Figure A, showing the bacteria shown in Table 6 spread on each plate. 5 shows the lytic activity of the fifth bacteriophage obtained in Example 5. The upper rows (1 to 3) of FIG. 5 show the lytic activity of the bacteriophage of SEQ ID NO: 18, and the lower rows (4 to 6) show the lytic activity of the bacteriophage of SEQ ID NO: 19. Panel A shows agar plates on which Xanthomonas bacteria shown in Table 7 and control Pseudomonas fluorescens were cultured, followed by dropping the fifth purified phage solution onto the plates and allowing them to stand. Panel B shows a plate diagram corresponding to Panel A, indicating the position of the dropped phage purified solution on each plate. In FIGS. 5A and 5B, panels 1 and 4 show plates on which bacteria with an ID of MAFF No. 211971 were spread, panels 2 and 5 show plates on which bacteria with an ID of MAFF No. 311351 were spread, and panels 3 and 6 show plates on which control Pseudomonas fluorescens with an ID of NCIMB-ID 10460 was spread. 1 shows the lytic activity of the sixth bacteriophage obtained in Example 6. FIG. 1A shows a diagram of Xanthomonas bacteria and control Pseudomonas fluorescens cultured on an agar plate, followed by dropping the sixth phage purified solution onto the center of the plate and allowing it to stand for static culture. FIG. 1B shows a diagram of the plate corresponding to FIG. 1A, showing the bacteria shown in Table 9 cultured on each plate. FIG. 1C shows the lytic activity of the seventh bacteriophage obtained in Example 7.7A is a diagram showing agar plates containing Xanthomonas bacteria shown in Table 10 and control Pseudomonas fluorescens, which were cultured, and then the seventh phage purified solution was added dropwise to the plates and allowed to stand. FIG. 7B is a diagram of the plates corresponding to FIG. 7A, showing the positions of the phage purified solution added dropwise to each plate. In FIGS. 7A and B, 1 indicates a plate containing bacteria with an ID of MAFF No. 311351, 2 indicates a plate containing bacteria with an ID of MAFF No. 311622, 3 indicates a plate containing bacteria with an ID of MAFF No. 106642, and 4 indicates a plate containing control Pseudomonas fluorescens with an ID of NCIMB-ID 10460. In FIG. 7B, a indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 28, and b indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 29. This diagram shows the lytic activity of the eighth bacteriophage obtained in Example 8. (A) is a diagram showing Xanthomonas bacteria shown in Table 11 and control Pseudomonas fluorescens cultured on agar plates, and then the eighth purified phage solution was added dropwise to the plates and allowed to stand. (B) is a diagram of the plates corresponding to (A), showing the positions of the purified phage solution added dropwise to each plate. In Figures 8A and B, 1 is a plate on which bacteria with an ID of MAFF No. 211971 were spread, 2 is a plate on which bacteria with an ID of MAFF No. 311282 were spread, 3 is a plate on which bacteria with an ID of MAFF No. 301420 were spread, 4 is a plate on which bacteria with an ID of MAFF No. 301426 were spread, 5 is a plate on which bacteria with an ID of MAFF No. 311351 were spread, 6 is a plate on which bacteria with an ID of MAFF No. 311414 were spread, 7 is a plate on which bacteria with an ID of MAFF No. 311417 were spread, 8 is a plate on which bacteria with an ID of MAFF No. 311562 were spread, 9 is a plate on which bacteria with an ID of MAFF No. 311571 were spread, 10 is a plate on which bacteria with an ID of MAFF No. 311618 were spread, and 11 is a plate on which bacteria with an ID of MAFF No. 311622 bacteria was spread on the plate, and 12 indicates a plate on which the control Pseudomonas fluorescens with ID NCIMB-ID 10460 was spread.In Figure 8B, "a" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 32. This figure shows the lytic activity of the eighth bacteriophage obtained in Example 8. Figure A shows the results of culturing the Xanthomonas bacteria shown in Table 11 and the control Pseudomonas fluorescens on an agar plate, followed by dropping the eighth purified phage solution onto the plate and allowing it to stand. Figure B is a plate diagram corresponding to Figure A, showing the position of the purified phage solution dropped onto each plate. 9A and 9B, 1 indicates a plate on which bacteria with an ID of MAFF No. 211971 were spread, 2 indicates a plate on which bacteria with an ID of MAFF No. 311282 were spread, 3 indicates a plate on which bacteria with an ID of MAFF No. 301420 were spread, 4 indicates a plate on which bacteria with an ID of MAFF No. 301426 were spread, 5 indicates a plate on which bacteria with an ID of MAFF No. 311351 were spread, and 6 indicates a plate on which control Pseudomonas fluorescens with an ID of NCIMB-ID 10460 was spread. In FIG. 9B, b indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 33, c indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 34, d indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 35, and e indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 36. This figure shows the lytic activity of the bacteriophages obtained in Example 9. 1A is a diagram showing the lytic activity of bacteriophages obtained in Example 10. FIG. ...B is a diagram showing the lytic activity of bacteriophages obtained in Example 10. FIG. 1B is a diagram showing the lytic activity of bacteriophages obtained in Example 10. FIG. 1A is a diagram showing the lytic activity of bacteriophages obtained in Example 10. FIG. 1B is a diagram showing the lytic activity of bacteriophages obtained in Example 10.

[0023] Figure 1 shows an example of a plate for a plaque assay of a second bacteriophage obtained in Example 2. Figure 2 shows the lytic activity of a second bacteriophage obtained in Example 11. Figure 3A shows a diagram of an agar plate in which Xanthomonas bacteria shown in Table 4 and control Pseudomonas fluorescens were cultured, and then the second phage purified solution was added dropwise to the plate and allowed to stand. Figure 4B shows a plate corresponding to Figure 3A, showing the position of the phage purified solution added dropwise to each plate. 14A and 14B, 1 indicates a plate on which bacteria with an ID of MAFF No. 673005 were spread, 2 indicates a plate on which bacteria with an ID of MAFF No. 301256 were spread, 3 indicates a plate on which bacteria with an ID of MAFF No. 301352 were spread, 4 indicates a plate on which bacteria with an ID of MAFF No. 212146 were spread, 5 indicates a plate on which bacteria with an ID of MAFF No. 311351 were spread, and 6 indicates a plate on which control Pseudomonas fluorescens with an ID of NCIMB-ID 10460 was spread. In FIG. 14B, a indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 47, and b indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 48. This figure shows the lytic activity of the seventh phage obtained in Example 2 and the two second bacteriophages obtained in Example 11. FIG. 1A is a diagram showing a Xanthomonas bacterium shown in Table 14 spread on an agar plate, followed by the addition of a second phage purified solution dropwise to the plate and static culture. FIG. 1B is a plate diagram corresponding to FIG. 1A, showing the position of the phage purified solution dropped onto each plate. In FIG. 1B, "a" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 12, "b" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 47, and "c" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 48. This shows the lytic activity of the seventh bacteriophage obtained in Example 12. FIG. 1A is a diagram showing a Xanthomonas bacterium shown in Table 10 spread on an agar plate, followed by the addition of a second phage purified solution dropwise to the plate and static culture. FIG. 1B is a plate diagram corresponding to FIG. 1A, showing the ID of the bacteria cultured on each plate.

[0033] Figure 1 shows the lytic activity of two types of seventh phages obtained in Example 7 and a phage obtained in Example 12. Figure 1A shows a diagram of Xanthomonas bacteria with the ID MAFF No. 301260 spread on an agar plate, followed by the addition of a drop of purified phage solution to the plate and static incubation. Figure 1B shows a diagram of the plate corresponding to Figure 1A, indicating the position of the purified phage solution added to each plate. In Figure 1B, "a" indicates the position of the purified phage solution having the genomic DNA sequence of SEQ ID NO: 28, "b" indicates the position of the purified phage solution having the genomic DNA sequence of SEQ ID NO: 29, and "c" indicates the position of the purified phage solution having the genomic DNA sequence of SEQ ID NO: 53 obtained in Example 12. Figure 1B shows the lytic activity of a ninth bacteriophage obtained in Example 13. Figure 1A shows a diagram of Xanthomonas bacteria and purified phage solution spread on an agar plate as shown in Table 13, added dropwise to the plate and static incubation. Figure 1B shows a diagram of the plate corresponding to Figure 1A, indicating the ID of the bacteria cultured on each plate. 1 shows the bacteriolytic activity of the bacteriophage combinations tested in Example 14. A shows a diagram of Xanthomonas bacteria with the ID MAFF No. 311351 cultured on an agar plate, followed by adding a drop of purified phage solution to the plate and allowing it to stand. B shows a diagram of the plate corresponding to A, showing the types of phages contained in the purified phage solution added to each plate. In Figure B, a represents the first phage having the genomic DNA sequence of SEQ ID NO:8, b represents the second phage having the genomic DNA sequence of SEQ ID NO:13, c represents the seventh phage having the genomic DNA sequence of SEQ ID NO:28, d represents the ninth phage having the genomic DNA sequence of SEQ ID NO:41, e represents the tenth phage having the genomic DNA sequence of SEQ ID NO:44, f represents the third phage having the genomic DNA sequence of SEQ ID NO:14, g represents the fourth phage having the genomic DNA sequence of SEQ ID NO:17, h represents the fifth phage having the genomic DNA sequence of SEQ ID NO:18, i represents the sixth phage having the genomic DNA sequence of SEQ ID NO:23, and j represents the eighth phage having the genomic DNA sequence of SEQ ID NO:32. In the figure, " / " indicates that the phages before and after it are used in combination. For example, "a / b" indicates that the first phage and the second phage are used in combination.1 shows the bacteriolytic activity of the bacteriophage combinations tested in Example 14. A shows a diagram of Xanthomonas bacteria with the ID MAFF No. 311351 cultured on an agar plate, followed by adding a drop of purified phage solution to the plate and allowing it to stand. B shows a diagram of the plate corresponding to A, showing the types of phages contained in the purified phage solution added to each plate. In Figure B, a represents the first phage having the genomic DNA sequence of SEQ ID NO:8, b represents the second phage having the genomic DNA sequence of SEQ ID NO:13, c represents the seventh phage having the genomic DNA sequence of SEQ ID NO:28, d represents the ninth phage having the genomic DNA sequence of SEQ ID NO:41, e represents the tenth phage having the genomic DNA sequence of SEQ ID NO:44, f represents the third phage having the genomic DNA sequence of SEQ ID NO:14, g represents the fourth phage having the genomic DNA sequence of SEQ ID NO:17, h represents the fifth phage having the genomic DNA sequence of SEQ ID NO:18, i represents the sixth phage having the genomic DNA sequence of SEQ ID NO:23, and j represents the eighth phage having the genomic DNA sequence of SEQ ID NO:32. In the figure, " / " indicates that the phages before and after it are used in combination. For example, "f / g" indicates that the third phage and the fourth phage are used in combination. This graph shows the results of a disease control efficacy test for peach bacterial bore tested in Example 15. The average disease incidence is shown as a relative value to the untreated group. The numbers below each bar indicate the type of phage used. In each experimental group, phages 1 to 8 were used alone. This graph shows the results of a disease control efficacy test for bacterial borre of peach tested in Example 15. The average disease incidence is shown as a relative value to the untreated group. The numbers below each bar indicate the type of phages used in combination. In each experimental group, a combination of four phages consisting of phages 1, 3, 5, and 7; a combination of four phages consisting of phages 2, 4, 6, and 8; and a combination of eight phages consisting of phages 1 to 8 were used. This graph shows the results of a disease control efficacy test for black rot of broccoli tested in Example 16.The average disease incidence is shown as a relative value to the untreated group. The value in each bar graph indicates the type of phage used. In the figure, a circle indicates that the phage was used, and a "-" indicates that the phage was not used. Φ1 indicates the first phage having the genomic DNA sequence of SEQ ID NO: 10, Φ2-1 indicates the second phage having the genomic DNA sequence of SEQ ID NO: 13, Φ2-2 indicates the second phage having the genomic DNA sequence of SEQ ID NO: 47, Φ7-1 indicates the seventh phage having the genomic DNA sequence of SEQ ID NO: 28, Φ7-2 indicates the seventh phage having the genomic DNA sequence of SEQ ID NO: 53, Φ9 indicates the ninth phage having the genomic DNA sequence of SEQ ID NO: 41, and Φ10 indicates the tenth phage having the genomic DNA sequence of SEQ ID NO: 44. This graph shows the results of a disease control efficacy test for bacterial spot of tomato tested in Example 17. The average disease incidence is shown as a relative value to the untreated group. The value in each bar graph indicates the type of phage used. In the figure, a circle indicates that the phage was used, and a "-" indicates that the phage was not used. Φ1 indicates the first phage having the genomic DNA sequence of SEQ ID NO: 10, Φ2-1 indicates the second phage having the genomic DNA sequence of SEQ ID NO: 13, Φ2-2 indicates the second phage having the genomic DNA sequence of SEQ ID NO: 47, Φ2-3 indicates the second phage having the genomic DNA sequence of SEQ ID NO: 48, Φ7 indicates the seventh phage having the genomic DNA sequence of SEQ ID NO: 28, and Φ10 indicates the tenth phage having the genomic DNA sequence of SEQ ID NO: 44.

[0028] 1. Lytic Agent 1-1. Overview A first aspect of the present invention is a lytic agent. The lytic agent of the present invention comprises a bacteriophage having a genome sequence including a specific base sequence.

[0029] The bacteriolytic agent of the present invention exhibits bacteriolytic activity against target bacteria that may be pathogenic bacteria causing plant diseases.

[0030] 1-2. Definitions Terms used in this specification are defined below.

[0031] As used herein, the term "lytic agent" refers to an agent consisting of a bacteriophage that has lytic activity against target bacteria.

[0032] "Bacteria" is one of the major lineages of organisms that divides the entire living kingdom into three parts, along with archaea and eukaryotes. Bacteria are composed of cells without a nucleus and can self-replicate if they have a source of nutrition. Based on the International Code of Bacteriological Nomenclature, bacteria are named by family, genus, and species.

[0033] As used herein, the term "target bacterium" refers to a host bacterium that can be targeted by the phage constituting the bacteriolytic agent of the present invention, or the phage contained in the composition and plant disease control composition of the present invention. Specifically, for example, the bacterium has a membrane surface receptor on its outer cell membrane that is recognized by the phage. Alternatively, for example, the bacterium has a membrane surface receptor on its outer cell membrane that is recognized by a tail fiber protein consisting of a specific amino acid sequence. Alternatively, the bacterium has a membrane surface receptor on its outer cell membrane that is recognized by a tail tube protein consisting of a specific amino acid sequence. The "membrane surface receptor" is the site where, for example, the tail and tail fibers of the phage bind, and is composed of proteins, lipopolysaccharides, pili, etc. present in the outer layer of the bacterial outer membrane. Specific examples of target bacteria in this specification include Xanthomonas bacteria.

[0034] "Xanthomonas bacteria" are bacteria belonging to the genus Xanthomonas. Xanthomonas bacteria generally produce a yellow pigment called xanthomonadin, and many of them are known to be plant pathogens. Classifications below species include subtypes and pathovar, which are indicated by adding subsp. or pv. after the bacterial name, respectively. The smallest unit of classification is the strain, which refers to a group of cells that are considered to be genetically uniform. Table 1 below shows representative Xanthomonas bacteria, their host plants, and the plant diseases they cause.

[0035]

[0036] Among Xanthomonas bacteria, Xanthomonas arboricola, Xanthomonas campestris, Xanthomonas citri, Xanthomonas oryzae, and Xanthomonas cucurbitae are preferred as target bacteria of the present invention, but are not limited thereto. Specific examples of Xanthomonas arboricola include Xanthomonas arboricola pv. pruni, which has the pathotype pruni, and Xanthomonas arboricola pv. juglandis, which has the pathotype juglandis. Specific examples of Xanthomonas campestris include Xanthomonas campestris pv. vesicatoria, which has the pathotype vesicatoria, Xanthomonas campestris pv. campestris, which has the pathotype campestris, Xanthomonas campestris pv. vitians, which has the pathotype vitians, and Xanthomonas campestris pv. raphani, which has the pathotype raphani. Specific examples of Xanthomonas citri include Xanthomonas citri subsp. citri. Specific examples of Xanthomonas oryzae include Xanthomonas oryzae pv. oryzae, which has the pathotype oryzae.

[0037] "Bacteriophage" (often abbreviated simply as "phage" herein, as mentioned above) is a general term for viruses that infect bacteria. A typical phage consists of three parts: a head, a tail, and tail fibers. The head is composed of a capsomere, a protein coat, and an icosahedral capsid (viral shell), encapsulating the phage's genomic DNA within its internal space. The tail has a tubular structure composed of a tail tube protein and a sheath protein covering it. One end of the tail is connected to the head, and the other end is connected to the tail fibers. The tail functions as an introduction tube, injecting the genomic DNA from the head into the host bacterial cell. The tail fibers are composed of several fibrous structures composed of tail fiber proteins. The tail and tail fibers perform host recognition and adsorption functions by recognizing receptors present on the outer membrane surface of the host bacterial cell and adsorbing to the cell surface. Phages have extremely high host specificity, and their characteristics are based on the function of their tails and tail fibers.

[0038] Because 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. In the lysogenic cycle, phages integrate their own DNA into the bacterial chromosome without lysing the target bacterium, and grow along with the growth of the bacterium. In the lytic cycle, phages self-multiply within the host bacterial cells, then lyse the host bacterium and release large amounts of progeny phages. The phages of the present invention are virulent phages that undergo the lytic cycle.

[0039] As described above, a "tail fiber protein" is a protein that constitutes the tail fiber of a phage. It is known that tail fiber proteins play an important role in the specificity of the host recognition and adsorption ability of the tail and tail fiber (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16:760-773). Therefore, even if the host bacterium is the same as that of a known phage, novel phages with characteristic tail fiber proteins have a different host recognition site, making them highly useful, as they can exhibit bacteriolytic activity even against bacteria that are resistant to infection by known phages.

[0040] The term "tail fiber gene" refers to a gene contained in the genomic DNA of a phage and encoding the tail fiber protein.

[0041] As mentioned above, "tail tube proteins" are proteins that make up the tubular structure of the phage tail. They interact with the tail fibers and, together with the tail fibers, play an important role in the specificity of host recognition and adsorption (Maozhi Hu, et al., 2020, 9:1, 855-867). Known tail tube proteins include tail tube fiber protein A and tail tube protein B. "Tail tube protein A" is a protein that forms a ring at the bottom of the tubular structure of the tail and interacts with the tail fibers. "Tail tube protein B" is a protein that forms the lower end of the tubular structure of the tail and binds to a receptor present on the outer membrane surface of the host bacterium.

[0042] The term "tailtube gene" refers to a gene contained in the phage genomic DNA that encodes the tailtube protein. The term "tailtube protein A gene" refers to the gene encoding tailtube protein A, and the term "tailtube protein B gene" refers to the gene encoding tailtube protein B.

[0043] "Bacteriolysis" refers to the phenomenon of destroying the bacterial cell membrane. As mentioned above, this phenomenon is mainly seen in the infection mode of virulent phages. Bacterial lysis results in the death of the bacteria. Bacteriolysis begins when the phage specifically adsorbs to the target bacterium and injects its own DNA into the target bacterium's cell via its tail. The phage then uses the bacterial translation mechanism to replicate itself and produce a large number of progeny phages, which then lyse the bacterium and release them into the outside world.

[0044] As used herein, "plant disease" refers to a general term for diseases that occur in plants. Known plant diseases include diseases caused by infectious pathogens such as viruses, bacteria, filamentous fungi, actinomycetes, viroids, phytoplasmas, nematodes, mites, or insects, as well as diseases caused by non-infectious pathogens such as a lack or excess of nutrients or water, or chemical damage. Unless otherwise specified, plant diseases in this specification refer to diseases caused by bacteria, i.e., plant pathogenic bacteria. Unless otherwise specified, plant pathogenic bacteria in this specification refer to the aforementioned target bacteria, for example, bacteria of the genus Xanthomonas.

[0045] As used herein, "control" refers to prevention or treatment (eradication) (from the website of the Japan Agricultural Chemicals Association). Therefore, as used herein, "plant disease control" refers to the prevention of plant diseases, particularly target bacteria, or the treatment of plant diseases caused by target bacteria.

[0046] As used herein, the term "target plant" refers to a plant to which the plant disease control composition of the present invention described below is applied. This plant corresponds to a plant that has developed a specific plant disease due to infection with the target bacterium, or a plant that is at risk of infection with the target bacterium.

[0047] 1-3. Constitution The lytic agent of the present invention comprises a bacteriophage.

[0048] <First Phage> The first phage is characterized by containing in its genomic DNA a gene encoding a tail fiber protein consisting of a specific amino acid sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0049] (1-1) Tail fiber protein The tail fiber protein consists of the amino acid sequence shown in SEQ ID NO: 1, which is composed of 1,371 amino acid residues, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 1. All of the tail fiber proteins are characterized by having target bacterium-specific recognition activity.

[0050] As used herein, "multiple" refers to 2 to 10 amino acids, for example, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Furthermore, "(amino acid) substitution" refers to substitutions within conservative amino acid groups that have similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples of such substitutions include substitutions within uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of the polypeptide.

[0051] Furthermore, as used herein, "amino acid sequence identity" refers to a numerical value indicating the percentage of sites with identical amino acid residue types within the comparison range of two amino acid sequences. Amino acid sequence identity can be calculated by aligning two amino acid sequences to maximize amino acid identity within the comparison range, even when the two sequences are different in length. A representative algorithm for such analysis is BLAST, although not limited thereto. BLAST is available in a variety of software and web services. For example, amino acid sequence identity can be easily calculated using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ) or the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). In addition to BLAST, there are also algorithms such as FASTA, which can be used if they can calculate reasonable identity.

[0052] The present inventors discovered three phages that have lytic activity against Xanthomonas bacteria and identified the tail fiber genes from the genomic DNA sequences of these phages (SEQ ID NOS: 8 to 10, respectively). Analysis of the amino acid sequences encoded by these tail fiber genes (SEQ ID NOS: 2 to 4) using the genetic information processing software GENETYX revealed that they share a high sequence identity of 98% or more. SEQ ID NOS: 1 is the amino acid sequence common to the amino acid sequences of SEQ ID NOS: 2 to 4.

[0053] Therefore, the amino acid sequence shown in SEQ ID NO:1 can be any of the amino acid sequences shown in SEQ ID NOs:2-4.

[0054] (1-2) Tail Fiber Gene The first bacteriophage contains a tail fiber gene consisting of a nucleotide sequence encoding the tail fiber protein in the phage genomic DNA.

[0055] Specific base sequences of the tail fiber gene include, for example, the base sequence shown in any one of SEQ ID NOs: 5 to 7, or a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in any one of SEQ ID NOs: 5 to 7, a base sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence shown in any one of SEQ ID NOs: 5 to 7, or a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in any one of SEQ ID NOs: 5 to 7.

[0056] As used herein, "nucleotide sequence identity" refers to a numerical value indicating the proportion of sites with the same type of base within the comparison range of two nucleotide sequences, similar to the amino acid sequence identity. Even if the lengths of the two nucleotide sequences are different, the nucleotide sequence identity can be calculated by aligning the sequences so that the degree of base identity within the comparison range is maximized. Analysis of nucleotide sequence identity can also be performed using, but is not limited to, the aforementioned BLAST and FASTA, as well as analysis algorithms such as MUMmer.

[0057] As used herein, the term "highly stringent conditions" refers to environmental conditions that make nonspecific hybridization unlikely. Under highly stringent conditions, a nucleic acid having a target nucleotide sequence can form a hybrid, but a nucleic acid having a nonspecific nucleotide sequence cannot substantially form a hybrid. Generally, highly stringent conditions refer to conditions with a low salt concentration and a high temperature. A low salt concentration is, for example, 15 to 750 mM, preferably 15 to 500 mM, 15 to 300 mM, or 15 to 200 mM. A high temperature is, 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.

[0058] The proteins encoded by the tail fiber genes all have bacteriolytic activity against target bacteria.

[0059] (1-3) Genomic DNA The first bacteriophage contains the tail fiber gene. There are no limitations on the genes or base sequences other than the tail fiber gene. The phage genomic DNA may be, for example, a nucleotide sequence shown in any one of SEQ ID NOs: 8 to 10 (201015 bp, 200185 bp, and 200277 bp, respectively), a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence of any one of SEQ ID NOs: 8 to 10 in a region other than the tail fiber gene, or a nucleotide sequence in which the nucleotide sequence of the region other than the tail fiber gene in the nucleotide sequence of any one of SEQ ID NOs: 8 to 10 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 of any one of SEQ ID NOs: 8 to 10. a base sequence having one or more bases added, deleted, and / or substituted in the base sequence shown in any one of SEQ ID NOs: 8 to 10, and a base sequence having a sequence identity of 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% or more when aligned with the base sequence shown in any one of SEQ ID NOs: 8 to 10, Examples of such genomic DNA include genomic DNAs consisting of a base sequence with 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.

[0060] Depending on the software and analysis server used herein, indexes of sequence identity may be expressed using, for example, average nucleotide identity (ANI), and these may also be used. It is not easy to align and compare the entire range of long phage genomic DNA. Therefore, the above-mentioned sequence identity may be achieved within the range automatically aligned by the software or web service. For example, in analysis using the NCBI-provided BLAST server, the maximum possible alignment is automatically performed, and the proportion of the range to be compared to the entire range of the phage genomic DNA is calculated as a value called Query Cover. When a partial range is aligned and sequence identity is calculated in this manner, the sequence identity of the base sequence across the entire range of the phage genomic DNA (entire range sequence identity) can also be estimated based on the results. For example, the Query Cover value can be multiplied by the sequence identity value within the aligned and compared range (sequence identity within the aligned range) to obtain an estimate of the entire range of sequence identity. In this case, further corrections, such as incorporating expected sequence identity in ranges outside the aligned range, may be made to improve the accuracy of the estimate.

[0061] For example, when BLAST analysis was performed using GENETYX-NGS implemented in GENETYX, the sequence identity (Average Nucleotide Identity: ANI) of SEQ ID NO: 8 to SEQ ID NO: 9 was 98% or more over 96% or more of the entire length, and the sequence identity to SEQ ID NO: 10 was 98% or more over 95% or more of the entire length. Therefore, the sequence identity of the genomic DNA sequence of SEQ ID NO: 8 to the entire length of the genomic DNA sequence of SEQ ID NO: 9 can be estimated to be 94% or more. Furthermore, the sequence identity of the genomic DNA sequence of SEQ ID NO: 8 to the entire length of the genomic DNA sequence of SEQ ID NO: 10 can be estimated to be 93% or more.

[0062] In this specification, phage genomic DNA packaging can be either linear or circular. Furthermore, in next-generation genome sequencer analysis, the genomic DNA is fragmented, the base sequences of the individual fragments are read, and the sequence is determined through analysis that connects them. In the case of phage, the sequence is often connected without a reference genomic DNA sequence (de novo assembly). Therefore, it is difficult to unambiguously determine the start and end of the analyzed genome (Merrill, B.D., et al. BMC Genomics, 2016 17, 679). The start and end of the genome sequence to be compared can be different, and this is automatically taken into account in analysis using software or analysis servers.

[0063] (1-4) Effects The lytic agent of the present invention containing the first phage can exhibit lytic activity against a wide range of bacterial species of the genus Xanthomonas, and can be applied to various plant diseases.

[0064] Generally, if a phage has too high specificity, it cannot cover the diversity of target bacteria, resulting in limited effectiveness. Furthermore, from an industrial perspective, it is preferable that the phage be applicable to multiple plant diseases. Therefore, phages that exhibit broad lytic activity against various bacterial species, such as the lytic agent of the present invention, are extremely useful.

[0065] <Second Phage> The second phage is characterized by containing in its genomic DNA a gene encoding a tail fiber protein consisting of a specific amino acid sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0066] (2-1) Tail Fiber Protein The tail fiber protein consists of the amino acid sequence shown in SEQ ID NO: 11, which is composed of 487 amino acid residues, or an amino acid sequence obtained by substituting one amino acid at positions other than 278 and 350 in the amino acid sequence shown in SEQ ID NO: 11. A tail fiber protein consisting of the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof can achieve extremely useful host specificity, being specific to bacteria of a specific genus and exhibiting bacteriolytic activity against a wide range of bacterial species within that specific genus.

[0067] As described above, when one amino acid is substituted in the amino acid sequence shown in SEQ ID NO: 11, the position is not particularly limited as long as it is a position other than positions 278 and 350. For example, one amino acid may be substituted at any amino acid in the range of positions 1 to 250 in the amino acid sequence shown in SEQ ID NO: 11. Specifically, for example, one amino acid may be substituted at positions 50 or later, 55 or later, 60 or later, 65 or later, 66 or later, 67 or later, 68 or later, 70 or later, 80 or later, 90 or later, 100 or later, 110 or later, 120 or later, 130 or later, 140 or later, 145 or later, 150 or later, 151 or later, 152 or later, 153 or later, or 154 or later in the amino acid sequence shown in SEQ ID NO: 11. Furthermore, for example, one amino acid substitution may be made at positions 200 or earlier, 190 or earlier, 180 or earlier, 170 or earlier, 160 or earlier, 159 or earlier, 158 or earlier, 157 or earlier, 156 or earlier, 155 or earlier, or 154 or earlier in the amino acid sequence shown in SEQ ID NO: 11. For example, one amino acid substitution may be made at positions 50 to 200, 67 to 156, 80 to 156, 100 to 156, 110 to 156, 120 to 156, 130 to 156, 140 to 156, 150 to 156, 153 to 156, 154 to 156, or 154. The amino acid region from positions 67 to 156 in the amino acid sequence shown in SEQ ID NO: 11 is thought to form one domain. Therefore, if one amino acid is substituted, any amino acid within this amino acid region may have the properties of the second phage, regardless of its position.

[0068] The type of amino acid substitution is not particularly limited. For example, it may be a conservative substitution, or may be a substitution within an amino acid group with a low polarity side chain (Gly, Asn, Gln, Ser, Thr, Cys, Tyr, Leu, Val, Ile, Val, Ala, Met, Pro). For example, it may be a substitution between an uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) or a neutral amino acid group with a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys) and a neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro). More specifically, it may be a substitution between threonine (Thr) and alanine (Ala). An example of such a substituted amino acid sequence is the amino acid sequence shown in SEQ ID NO: 45. In this sequence, the amino acid at position 154 in the amino acid sequence shown in SEQ ID NO: 11 is substituted from threonine (Thr) to alanine (Ala).

[0069] (2-2) Tail Fiber Gene The second bacteriophage contains a tail fiber gene consisting of a nucleotide sequence encoding the tail fiber protein in the phage genomic DNA.

[0070] The specific base sequence of the tail fiber gene is not particularly limited, as long as it encodes the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence in which one amino acid other than that at positions 278 and 350 in the amino acid sequence shown in SEQ ID NO: 11 has been substituted. For example, it refers to a gene consisting of the base sequence shown in SEQ ID NO: 12, which encodes the amino acid sequence shown in SEQ ID NO: 11. Another example is a gene consisting of the base sequence shown in SEQ ID NO: 46, which encodes the amino acid sequence shown in SEQ ID NO: 45.

[0071] The protein encoded by the tail fiber gene can achieve extremely useful host specificity, which is specific to a specific genus of bacteria and exhibits bacteriolytic activity against a wide range of bacterial species within that specific genus.

[0072] (2-3) Genomic DNA The second bacteriophage contains the tail fiber gene. There are no limitations on the genes or base sequences other than the tail fiber gene. For example, the phage genomic DNA may be a 63,753 bp nucleotide sequence shown in SEQ ID NO: 13, a 63,743 bp nucleotide sequence shown in SEQ ID NO: 47 or 48, a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence of SEQ ID NO: 13, 47, or 48 in a region other than the tail fiber gene, or a nucleotide sequence in which the nucleotide sequence of the region other than the tail fiber gene in the nucleotide sequence of SEQ ID NO: 13, 47, or 48 is identical to the nucleotide sequence of SEQ ID NO: 13, 47, or 48 by 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% 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. Examples of such genomic DNA include a base sequence having 7% or more, 99.8% or more, or 99.9% or more sequence identity; a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 13, 47, or 48; a base sequence having 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 sequence identity when aligned with the base sequence shown in SEQ ID NO: 13, 47, or 48; and a base sequence that hybridizes under highly stringent conditions to the base sequence in a region of the base sequence shown in SEQ ID NO: 13, 47, or 48 other than the tail fiber gene.

[0073] (2-4) Effects The lytic agent of the present invention containing the second phage can exhibit lytic activity against a wide range of Xanthomonas species and can be applied to various plant diseases.

[0074] Generally, if a phage is too specific, it will not be able to cover the diversity of target bacteria, resulting in limited effectiveness. Furthermore, from an industrial perspective, it is preferable that the phage be applicable to multiple plant diseases. Therefore, phages that exhibit broad lytic activity against a variety of bacterial species, such as the lytic agent of the present invention containing the second phage, are extremely useful.

[0075] <Third Phage> The third phage is characterized by having a genomic DNA sequence containing a specific base sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0076] (3-1) Genomic DNA The genomic DNA sequence comprises or consists of the 61,291 bp nucleotide sequence shown in SEQ ID NO: 14, a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 14, or a nucleotide sequence having 80% or more, 83% or more, 85% or more, 88% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 14. Bacteriophages having any of these genomic DNA sequences are characterized by being able to specifically adsorb to target bacteria and inject their genomic DNA into the cells of the target bacteria.

[0077] (3-2) Effects The lytic agent of the present invention containing the third phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be used to treat plant diseases.

[0078] <Fourth Phage> The fourth phage is characterized by containing in its genomic DNA a gene encoding a tail fiber protein consisting of a specific amino acid sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0079] (4-1) Tail Fiber Protein The tail fiber protein comprises the amino acid sequence shown in SEQ ID NO: 15, which is composed of 604 amino acid residues, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 15, 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 amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 15. All of the tail fiber proteins are characterized by having target bacterium-specific recognition activity.

[0080] (4-2) Tail Fiber Gene The fourth bacteriophage contains a tail fiber gene consisting of a nucleotide sequence encoding the tail fiber protein in the phage genomic DNA.

[0081] Specific examples of the base sequence of the tail fiber gene include the base sequence shown in SEQ ID NO: 16, which encodes the amino acid sequence shown in SEQ ID NO: 15, or a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 16, or a base sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more base sequence identity with the base sequence shown in SEQ ID NO: 16, or a gene consisting of a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 16.

[0082] The proteins encoded by the tail fiber genes all have bacteriolytic activity against target bacteria.

[0083] (4-3) Genomic DNA The fourth bacteriophage contains the tail fiber gene. The genes and nucleotide sequences other than the tail fiber gene are not limited. For example, the phage genomic DNA may be the 46,393 bp nucleotide sequence shown in SEQ ID NO: 17, the nucleotide sequence shown in SEQ ID NO: 3 in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence in the region other than the tail fiber gene, the nucleotide sequence shown in SEQ ID NO: 17 in which the nucleotide sequence in the region other than the tail fiber gene 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 to the nucleotide sequence shown in SEQ ID NO: 17, or the salt of the phage shown in SEQ ID NO: 17. Examples of such genomic DNA include a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence, and a base sequence that has a sequence identity of 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% or more, 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 when aligned with the base sequence shown in SEQ ID NO: 17.

[0084] (4-4) Effects The lytic agent of the present invention containing the fourth phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be used to treat plant diseases.

[0085] <Fifth Phage> The fifth phage is characterized by having a genomic DNA sequence containing a specific base sequence, and the lytic agent of the present invention exhibits specific lytic activity against target bacteria.

[0086] (5-1) Genomic DNA The fifth bacteriophage has a genomic DNA sequence of the phage that is the 43,177 bp nucleotide sequence shown in SEQ ID NO: 18 or the 43,741 bp nucleotide sequence shown in SEQ ID NO: 19, or a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 18 or 19, or a nucleotide sequence that has 90% 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 SEQ ID NO: 18 or 19, or a nucleotide sequence that is aligned with the nucleotide sequence shown in SEQ ID NO: 18 or 19. When arranged, the base sequence has a 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, and further has a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 18 or 19.

[0087] (5-2) Effects The lytic agent of the present invention containing the fifth phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be used to treat plant diseases.

[0088] <Sixth phage> The sixth phage is characterized by containing in its genomic DNA a gene encoding the protein newly discovered in the present invention (often simply referred to as the "protein of the present invention" in this specification), and exhibits specific bacteriolytic activity against target bacteria.

[0089] (6-1) Protein of the Present Invention The protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 21, which is composed of 84 amino acid residues, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 21, 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 amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 21. All of the proteins of the present invention are characterized by having target bacterium-specific recognition activity.

[0090] The protein of the present invention is a novel protein that plays an important role in determining the host range.

[0091] (6-2) Gene encoding the protein of the present invention The sixth bacteriophage contains the gene encoding the protein of the present invention (often simply referred to herein as the "gene of the present invention") in the genomic DNA of the phage.

[0092] The gene of the present invention is not particularly limited as long as it is a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO: 21. Specific examples of the nucleotide sequence include the nucleotide sequence shown in SEQ ID NO: 22 that encodes the amino acid sequence shown in SEQ ID NO: 21, or the nucleotide sequence shown in SEQ ID NO: 22 in which one or more nucleotides have been added, deleted, and / or substituted, or a nucleotide sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 22, or a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 22.

[0093] The proteins encoded by the genes of the present invention all have bacteriolytic activity against target bacteria.

[0094] (6-3) Genomic DNA The sixth bacteriophage contains the gene of the present invention. There is no limitation on the genes and base sequences other than the gene of the present invention. For example, the phage genomic DNA may be a 42,708 bp base sequence shown in SEQ ID NO: 23, a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence of SEQ ID NO: 23 in a region other than the gene of the present invention, a base sequence in which the base sequence of SEQ ID NO: 23 in a region other than the gene of the present invention has 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence of SEQ ID NO: 23, a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence of SEQ ID NO: 23 in a region other than the gene of the present invention, or a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence of SEQ ID NO: 23 in a region other than the gene of the present invention has 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence of SEQ ID NO: 23, or a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence of SEQ ID NO: 23 or a nucleotide sequence in which multiple nucleotides have been added, deleted, and / or substituted, and further, a genomic DNA consisting of a nucleotide sequence that, when aligned with the nucleotide sequence shown in SEQ ID NO: 23, has 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.

[0095] (6-4) Effects The lytic agent of the present invention containing the sixth phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be used to treat plant diseases.

[0096] <Seventh Phage> The seventh phage is characterized by containing in its genomic DNA a gene encoding a tailtube protein consisting of a specific amino acid sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0097] (7-1) Tail tube protein The tail tube protein includes tail tube protein A and tail tube protein B. The tail tube protein A consists of the amino acid sequence shown in SEQ ID NO: 24 or 49, which is composed of 205 amino acid residues, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 24 or 49, or an amino acid sequence having 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 24 or 49.

[0098] The amino acid substitutions herein can include amino acid substitutions at one or more positions selected from the group consisting of positions 28, 108, 110, 153, 157, 189, and 201 in the amino acid sequence shown in SEQ ID NO: 24 or 49. For example, substitutions at two or more, three or more, four or more, five or more, six or more, or all of these positions can be included.

[0099] The specific type of amino acid substitution is not particularly limited. For example, it may be a conservative substitution or may include one or more non-conservative substitutions. Specifically, for example, the amino acid sequence of tailtube protein A of the seventh phage may be the amino acid sequence set forth in SEQ ID NO:60. This amino acid sequence is identical to the amino acid sequence set forth in SEQ ID NO:24 or 49 except for the amino acids at the following positions: glutamic acid (Glu) or aspartic acid (Asp) at position 28, serine (Ser) or asparagine (Asn) at position 108, glutamine (Gln) or histidine (His) at position 110, glutamine (Gln) or lysine (Lys) at position 153, aspartic acid (Asp) or asparagine (Asn) at position 157, tyrosine (Tyr) or phenylalanine (Phe) at position 189, and valine (Val) or tyrosine (Tyr) at position 201.

[0100] The tail tube protein B consists of an amino acid sequence of 834 amino acid residues shown in SEQ ID NO: 25 (correctly SEQ ID NO: 57) or 50, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 25 (correctly SEQ ID NO: 57) or 50, or an amino acid sequence having 97% or more, 97.5% or more, 97.6% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 25 (correctly SEQ ID NO: 57) or 50.

[0101] The amino acid substitutions herein can include amino acid substitutions at one or more positions selected from the group consisting of positions 25, 53, 216, 221, 272, 389, 395, 410, 425, 472, 607, 623, 662, 670, 699, 707, 757, 763, 764, and 765 in the amino acid sequence set forth in SEQ ID NO: 57 or 50. For example, substitutions at two or more, three or more, four or more, five or more, ten or more, fifteen or more, or all of these positions can be included.

[0102] The specific type of amino acid substitution is not particularly limited. For example, it may be a conservative substitution, or it may include one or several non-conservative substitutions. Specifically, for example, the amino acid sequence of the tailtube protein B of the seventh phage may be the amino acid sequence shown in SEQ ID NO:61. This amino acid sequence is identical to the amino acid sequence set forth in SEQ ID NO:57 or 50, except for the amino acids at the following positions: alanine (Ala) or proline (Pro) at position 25, alanine (Ala) or serine (Ser) at position 53, alanine (Ala) or proline (Pro) at position 216, histidine (His) or tyrosine (Tyr) at position 221, valine (Val) or (Ile) at position 272, alanine (Ala) or glutamic acid (Glu) at position 389, serine (Ser) or alanine (Ala) at position 395, valine (Val) or isoleucine (Ile) at position 410, isoleucine (Ile) or valine (Val) at position 425, and glutamic acid (Glu) at position 472. position 623 isoleucine (Ile) or valine (Val), position 662 is arginine (Arg) or serine (Ser), position 670 is leucine (Leu) or glutamine (Gln), position 699 is threonine (Thr) or asparagine (Asn), position 707 is aspartic acid (Asp) or glycine (Gly), position 757 is serine (Ser) or alanine (Ala), position 763 is glycine (Gly) or serine (Ser), position 764 is serine (Ser) or asparagine (Asn), and position 765 is methionine (Met) or valine (Val).

[0103] Both tailtube proteins are characterized by their involvement in target bacterium-specific recognition activity.

[0104] (7-2) Tailtube Gene The seventh bacteriophage contains a tailtube gene consisting of a base sequence encoding the tailtube protein in the phage genomic DNA.

[0105] The tail tube gene is not particularly limited as long as it is a gene that encodes a tail tube protein, and includes tail tube protein A gene and tail tube protein B gene.

[0106] Specific examples of the nucleotide sequence of the tail tube protein A gene include the nucleotide sequence shown in SEQ ID NO: 26 or 51, which encodes the amino acid sequence shown in SEQ ID NO: 24 or 49, or the nucleotide sequence shown in SEQ ID NO: 26 or 51 in which one or more nucleotides have been added, deleted, and / or substituted, or a nucleotide sequence that has 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more nucleotide identity to the nucleotide sequence shown in SEQ ID NO: 26 or 51, or a gene consisting of a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 26 or 51. The nucleotide sequence of the tail tube protein A gene may also be a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 60.

[0107] Specific examples of the nucleotide sequence of the tail tube protein B gene include the nucleotide sequence shown in SEQ ID NO: 27 (correctly SEQ ID NO: 58) or 52, which encodes the amino acid sequence shown in SEQ ID NO: 25 (correctly SEQ ID NO: 57) or 50, or a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 27 (correctly SEQ ID NO: 58) or 52, or a nucleotide sequence that has 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more nucleotide identity to the nucleotide sequence shown in SEQ ID NO: 27 (correctly SEQ ID NO: 58) or 52, or a gene consisting of a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 27 (correctly SEQ ID NO: 58) or 52. The nucleotide sequence of the tail tube protein B gene may also be a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 61.

[0108] The proteins encoded by the tailtube genes all have bacteriolytic activity against target bacteria.

[0109] (7-3) Genomic DNA The seventh bacteriophage contains the tailtube gene. The genes and nucleotide sequences other than the tailtube gene are not limited. For example, the phage genomic DNA may be the 44,716 bp nucleotide sequence shown in SEQ ID NO: 28, the 44,829 bp nucleotide sequence shown in SEQ ID NO: 29, or the 44,585 bp nucleotide sequence shown in SEQ ID NO: 53, or a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence in the region other than the tailtube gene in the nucleotide sequence shown in SEQ ID NO: 28, 29, or 53, or a nucleotide sequence in which the nucleotide sequence in the region other than the tailtube gene in the nucleotide sequence shown in SEQ ID NO: 28, 29, or 53 has 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 28, 29, or 53. Examples of such genomic DNA include a base sequence having the same sequence identity as the base sequence shown in SEQ ID NO: 28, 29, or 53, a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 28, 29, or 53, and a base sequence having 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 sequence identity when aligned with the base sequence shown in SEQ ID NO: 28, 29, or 53.

[0110] (7-4) Effects The lytic agent of the present invention containing the seventh phage can exhibit lytic activity against a wide range of bacterial species of the genus Xanthomonas, and can be applied to various plant diseases.

[0111] Generally, if a phage is too specific, it cannot cover the diversity of target bacteria, resulting in limited effectiveness. Furthermore, from an industrial perspective, it is preferable that the phage be applicable to multiple plant diseases. Therefore, phages that exhibit lytic activity against two or more bacterial species, such as the lytic agent of the present invention containing the seventh phage, are extremely useful.

[0112] <Eighth Phage> The eighth phage is characterized by having a genomic DNA sequence containing a specific base sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0113] (8-1) Genomic DNA The genomic DNA sequence includes a base sequence shown in any one of SEQ ID NOs: 32 to 36 (44388 bp, 44377 bp, 45279 bp, 44378 bp, and 44408 bp, respectively), a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in any one of SEQ ID NOs: 32 to 36, and a base sequence that is 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, 90.5% or more, 91. Examples of such genomic DNA sequences include genomic DNA sequences comprising or consisting of a nucleotide sequence having 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% or more, 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 sequence identity.

[0114] (8-2) Effects The lytic agent of the present invention containing the eighth phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be used to treat plant diseases.

[0115] <Ninth Phage> The ninth phage is characterized by containing in its genomic DNA a gene encoding a tailtube protein consisting of a specific amino acid sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0116] (9-1) Tail tube protein The tail tube protein includes tail tube protein A and tail tube protein B. The tail tube protein A consists of the amino acid sequence shown in SEQ ID NO: 37, which is composed of 206 amino acid residues, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 37.

[0117] The tail tube protein B comprises an amino acid sequence of 847 amino acid residues shown in SEQ ID NO: 38 or 54, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 38 or 54, or an amino acid sequence having 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, or 99.5% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 38 or 54.

[0118] The amino acid substitutions herein can include amino acid substitutions at one or more positions selected from the group consisting of positions 61, 108, 404, 679, 682, and 727 in the amino acid sequence shown in SEQ ID NO: 38 or 54. For example, substitutions at two or more, three or more, four or more, five or more, or all of these positions can be included.

[0119] The specific type of amino acid substitution is not particularly limited. For example, it may be a conservative substitution or may include one or several non-conservative substitutions. For example, it may be a substitution within an amino acid group with a low polar side chain (Gly, Asn, Gln, Ser, Thr, Cys, Tyr, Leu, Val, Ile, Val, Ala, Met, Pro). For example, it may be a substitution between an uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) or a neutral amino acid group with a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys) and a neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro). More specifically, it may be a substitution between threonine (Thr) and alanine (Ala).

[0120] A specific example of the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 59. This amino acid sequence is identical to the amino acid sequence shown in SEQ ID NO: 38, except for the amino acids at the following positions: the amino acid at position 61 is threonine (Thr) or alanine (Ala), the amino acid at position 108 is glutamine (Gln) or lysine (Lys), the amino acid at position 404 is proline (Pro) or glutamine (Gln), the amino acid at position 679 is proline (Pro) or serine (Ser), the amino acid at position 682 is threonine (Thr) or alanine (Ala), and the amino acid at position 727 is isoleucine (Ile) or valine (Val).

[0121] The ninth phage of the present invention also includes a phage having an amino acid sequence of tailtube protein B in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence that has 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, or 99.5% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:59.

[0122] Both tailtube proteins are characterized by their involvement in target bacterium-specific recognition activity.

[0123] (9-2) Tailtube Gene The ninth bacteriophage contains a tailtube gene consisting of a base sequence encoding the tailtube protein in the phage genomic DNA.

[0124] The tail tube gene is not particularly limited as long as it is a gene that encodes a tail tube protein, and includes tail tube protein A gene and tail tube protein B gene.

[0125] Specific examples of the nucleotide sequence of the tail tube protein A gene include the nucleotide sequence shown in SEQ ID NO: 39, which encodes the amino acid sequence shown in SEQ ID NO: 37, or the nucleotide sequence shown in SEQ ID NO: 39 in which one or more nucleotides have been added, deleted, and / or substituted, or a nucleotide sequence having 90% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more nucleotide identity to the nucleotide sequence shown in SEQ ID NO: 39, or a gene consisting of a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 39. The nucleotide sequence of the tail tube protein A gene may also be a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 59.

[0126] Specific examples of the nucleotide sequence of the tail tube protein B gene include the nucleotide sequence shown in SEQ ID NO: 40 or 55, which encodes the amino acid sequence shown in SEQ ID NO: 38 or 54, or the nucleotide sequence shown in SEQ ID NO: 40 or 55 in which one or more nucleotides have been added, deleted, and / or substituted, or a nucleotide sequence having 90% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more nucleotide identity to the nucleotide sequence shown in SEQ ID NO: 40 or 55, or a gene consisting of a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 40 or 55. Specific examples of the nucleotide sequence of the tail tube protein B gene include the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 59.

[0127] The proteins encoded by the tailtube genes all have bacteriolytic activity against target bacteria.

[0128] (9-3) Genomic DNA The ninth bacteriophage contains the tail tube gene. There are no limitations on the genes or base sequences other than the tail tube gene. For example, the phage genomic DNA may be a 43,667 bp nucleotide sequence shown in SEQ ID NO: 41 or a 43,336 bp nucleotide sequence shown in SEQ ID NO: 56, or a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence in a region other than the tailtube gene in the nucleotide sequence shown in SEQ ID NO: 41 or 56, or a nucleotide sequence in which the nucleotide sequence in the region other than the tailtube gene in the nucleotide sequence shown in SEQ ID NO: 41 or 56 is 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 similar to the nucleotide sequence shown in SEQ ID NO: 41 or 56. , a base sequence having 98% or more, or 99% or more sequence identity to the base sequence shown in SEQ ID NO: 41 or 56, a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 41 or 56, and further, genomic DNA consisting of a base sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 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 sequence identity when aligned with the base sequence shown in SEQ ID NO: 41 or 56.

[0129] (9-4) Effects The bacteriolytic agent of the present invention can exhibit bacteriolytic activity against a wide range of species of Xanthomonas, and can be applied to various plant diseases.

[0130] Generally, if a phage has too high specificity, it cannot cover the diversity of target bacteria, resulting in limited effectiveness. Furthermore, from an industrial perspective, it is preferable that the phage be applicable to multiple plant diseases. Therefore, phages that exhibit lytic activity against two or more bacterial species, such as the lytic agent of the present invention, are extremely useful.

[0131] <Tenth Phage> The tenth phage is characterized by containing in its genomic DNA a gene encoding a tail fiber protein consisting of a specific amino acid sequence, and exhibits specific bacteriolytic activity against target bacteria.

[0132] (10-1) Tail Fiber Protein The tail fiber protein comprises the amino acid sequence shown in SEQ ID NO: 42, which is composed of 568 amino acid residues, an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 42, 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 amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 42. All of the tail fiber proteins are characterized by having target bacterium-specific recognition activity.

[0133] (10-2) Tail Fiber Gene The tenth bacteriophage contains a tail fiber gene consisting of a nucleotide sequence encoding the tail fiber protein in the phage genomic DNA.

[0134] Specific examples of the base sequence of the tail fiber gene include the base sequence shown in SEQ ID NO: 43, which encodes the amino acid sequence shown in SEQ ID NO: 42, or a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 43, or a base sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more base sequence identity with the base sequence shown in SEQ ID NO: 43, or a gene consisting of a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 43.

[0135] The proteins encoded by the tail fiber genes all have bacteriolytic activity against target bacteria.

[0136] (10-3) Genomic DNA The tenth bacteriophage contains the tail fiber gene. There are no limitations on the genes or base sequences other than the tail fiber gene. For example, the phage genomic DNA may be a 76,340 bp nucleotide sequence shown in SEQ ID NO: 44, a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence of SEQ ID NO: 44 in a region other than the tail fiber gene, a nucleotide sequence in which the nucleotide sequence of the region other than the tail fiber gene in the nucleotide sequence of SEQ ID NO: 44 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 to the nucleotide sequence of SEQ ID NO: 44, a salt of SEQ ID NO: 44, or a salt of SEQ ID NO: 44. Examples of such genomic DNA include a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence, and a base sequence that, when aligned with the base sequence shown in SEQ ID NO: 44, has a sequence identity of 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% or more, 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.

[0137] (10-4) Effects The bacteriolytic agent of the present invention can exhibit bacteriolytic activity against a wide range of species of Xanthomonas, and can be applied to various plant diseases.

[0138] 2. Plant Disease Control Composition 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 lytic agent described in the first aspect as an active ingredient.

[0139] The composition of the present invention can 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 target plant diseases when used to control plant diseases.

[0140] In this specification, the term "plant disease control composition" refers to the case where the composition of the present invention is used for plant disease control.

[0141] 2-2. Composition 2-2-1. Constituent Components The composition of the present invention contains, as an active ingredient, the bacteriophage that is the lytic agent according to the first aspect as an essential component. It may also contain an agriculturally acceptable carrier and / or medium to the extent that it does not inhibit or suppress the lytic activity of the phage against target bacteria. If necessary, it may further contain other active ingredients. Each of the constituent components will be specifically described below.

[0142] (1) Active ingredient (lytic agent) The composition of the present invention contains, as an essential active ingredient, the lytic agent described in aspect 1. In the plant disease control composition, this active ingredient lyses the target bacterium of the present invention, thereby making it possible to prevent or treat plant diseases caused by the target bacterium.

[0143] The specific constitution of the lytic agent is described in detail in the first embodiment, and therefore will not be described here.

[0144] When used for plant disease control, the amount of active ingredient contained per unit amount in the composition depends on various conditions, such as the formulation, type of plant pathogenic bacterium, type of target plant, application location, and application method. It is preferable that the phage, which is the active ingredient, is contained in an amount sufficient to contact and infect plant pathogenic bacteria that have infected the target plant. Therefore, the amount of the lytic agent contained in the plant disease control composition of the present invention can be determined, taking into account various conditions, within the scope of common general technical knowledge in the field, so that the amount of the lytic agent contained in the plant disease control composition of the present invention is effective against the target bacteria after application.

[0145] The plant disease control composition of the present invention can contain, in combination as an active ingredient, one or more other phages that specifically recognize Xanthomonas bacteria and have lytic activity, as specifically exemplified below. For example, even if the target bacteria is the same, a synergistic or complementary effect of lytic activity can be expected by combining phages that recognize different cell surface receptors.

[0146] When the composition of the present invention contains multiple types of phages, the specific types of phages are not particularly limited. For example, the composition may contain only the phages described herein as active ingredients, or any other phages as additional active ingredients. Furthermore, the composition may contain phages with similar host ranges and / or phages with dissimilar host ranges. 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 genus), combinations of phages that commonly exhibit lytic activity against bacteria of one or more species of the same genus (e.g., Xanthomonas arboricola), and combinations of phages that commonly exhibit lytic activity against bacteria of one or more pathotypes of the same genus (e.g., Xanthomonas arboricola pv. pruni). Examples of combinations of phages with dissimilar host ranges include combinations of phages that exhibit lytic activity against bacteria belonging to a specific genus (e.g., Xanthomonas) and phages that do not; combinations of phages that exhibit lytic activity against bacteria of a specific pathotype of a specific genus (e.g., Xanthomonas arboricola pv. pruni) and phages that do not; combinations of phages that exhibit lytic activity against bacteria of a specific species of a specific genus (e.g., Xanthomonas arboricola) and phages that do not; and combinations of phages that exhibit specific lytic activity against bacteria of a specific genus (e.g., Xanthomonas), species (e.g., Xanthomonas arboricola), or pathotype (e.g., Xanthomonas arboricola pv. pruni) and phages that exhibit non-specific lytic activity.

[0147] For example, the composition of the present invention may contain a combination of two or more phages from the first to tenth phages described herein. Specifically, for example, the composition may contain a combination of two or more phages selected from the group consisting of the first phage, the second phage, the seventh phage, the ninth phage, and the tenth phage. Furthermore, for example, the composition may contain a combination of two or more phages selected from the group consisting of the third phage, the fourth phage, the fifth phage, the sixth phage, and the eighth phage. Furthermore, for example, the composition may contain a combination of one or more phages selected from the group consisting of the first phage, the second phage, the seventh phage, the ninth phage, and the tenth phage with one or more phages selected from the group consisting of the third phage, the fourth phage, the fifth phage, the sixth phage, and the eighth phage.

[0148] The number of phages (first to tenth) described herein contained in the composition of the present invention is not particularly limited, as long as it is one or more. For example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more phages may be included. Furthermore, the composition of the present invention may contain multiple phages that are within the scope of each phage (e.g., the second phage) but have different genomic DNA sequences. Specifically, the composition may contain two or more types of the second phage and / or two or more types of the seventh phage.

[0149] Examples include phages that contain in their genomic DNA a gene encoding the amino acid sequence shown in SEQ ID NO: 1 as an amino acid sequence common to tail fiber proteins, specifically phages that contain in their genomic DNA a gene encoding the amino acid sequence shown in any of SEQ ID NOs: 2 to 4, and phages that contain in their genomic DNA a gene encoding an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted, or an amino acid sequence with 90% or more sequence identity, to the amino acid sequence shown in any of SEQ ID NOs: 1 to 4. Specific examples of genes that encode such amino acid sequences include genes consisting of the nucleotide sequence shown in any of SEQ ID NOs: 5 to 7, and nucleotide sequences in which one or more bases have been added, deleted, and / or substituted, or a nucleotide sequence with 90% or more sequence identity, to the nucleotide sequence shown in any of SEQ ID NOs: 5 to 7. Examples of phage genomic DNA containing such genes include genomic DNA consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 8 to 10, and a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in any one of SEQ ID NOs: 8 to 10, a nucleotide sequence with 90% or more sequence identity, a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 2 to 4, or a genomic DNA consisting of a nucleotide sequence with 80% or more sequence identity to a nucleotide sequence other than the nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 2 to 4.

[0150] Other examples include phages that contain in their genomic DNA a gene encoding the amino acid sequence of SEQ ID NO: 11 or 45 as the amino acid sequence of the tail fiber protein, and phages that contain in their genomic DNA a gene encoding an amino acid sequence in which one or more amino acids other than those at positions 278 and 350 in the amino acid sequence of SEQ ID NO: 11 have been added, deleted, and / or substituted, or an amino acid sequence with 90% or more sequence identity. Specific examples of genes that encode such amino acid sequences include genes consisting of the nucleotide sequence of SEQ ID NO: 12 or 46, and nucleotide sequences in which one or more bases have been added, deleted, and / or substituted in the nucleotide sequence of SEQ ID NO: 12 or 46, or nucleotide sequences with 90% or more sequence identity. Furthermore, specific examples of genomic DNA of a phage containing such a gene include genomic DNA consisting of the base sequence shown in SEQ ID NO: 13, 47, or 48, and a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 13, 47, or 48, a base sequence with 90% or more sequence identity, a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 11 or 45, or a genomic DNA consisting of a base sequence with 80% or more sequence identity to a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 11 or 45.

[0151] Further examples of the phage genomic DNA include genomic DNA containing the nucleotide sequence shown in any one of SEQ ID NOs: 14, 18, 19, and 32 to 36, nucleotide sequences in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in any one of SEQ ID NOs: 14, 18, 19, and 32 to 36, and genomic DNA containing a nucleotide sequence having 90% or more sequence identity.

[0152] Other examples include phages that contain in their genomic DNA a gene encoding the amino acid sequence shown in SEQ ID NO: 15 or 42 as the amino acid sequence of the tail fiber protein, and phages that contain in their genomic DNA a gene encoding an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted, or an amino acid sequence with 90% or more sequence identity, to the amino acid sequence shown in SEQ ID NO: 15 or 42. Specific examples of genes that encode such amino acid sequences include genes consisting of the nucleotide sequence shown in SEQ ID NO: 16 or 43, and nucleotide sequences in which one or more bases have been added, deleted, and / or substituted, or a nucleotide sequence with 90% or more sequence identity, to the nucleotide sequence shown in SEQ ID NO: 16 or 43. Furthermore, specific examples of genomic DNA of a phage containing such a gene include, for example, genomic DNA consisting of the base sequence shown in SEQ ID NO: 17 or 44, and a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 17 or 44, a base sequence having 90% or more sequence identity, a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 15 or 42, or a genomic DNA consisting of a base sequence having 80% or more sequence identity with a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 15 or 42.

[0153] Further examples include a phage containing in its genomic DNA a gene encoding the amino acid sequence shown in SEQ ID NO: 21, and a phage containing in its genomic DNA a gene encoding an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted, or an amino acid sequence having 90% or more sequence identity, in the amino acid sequence shown in SEQ ID NO: 21. Specific examples of genes encoding such amino acid sequences include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 22, and a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted, or a nucleotide sequence having 90% or more sequence identity, in the nucleotide sequence shown in SEQ ID NO: 22. Furthermore, specific examples of genomic DNA of a phage containing such a gene include genomic DNA consisting of the base sequence shown in SEQ ID NO: 23, and a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 23, a base sequence having 90% or more sequence identity, a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 21, or a genomic DNA consisting of a base sequence having 80% or more sequence identity with a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 21.

[0154] Further examples include phages that contain in their genomic DNA a TTPA (Tail-Tubular protein A) gene consisting of the amino acid sequence shown in SEQ ID NO: 24, 49, 60 or 37 and a TTPB (Tail-Tubular protein B) gene consisting of the amino acid sequence shown in SEQ ID NO: 25 (correctly SEQ ID NO: 57), 50, 61, or 38, 54 or 59, and phages that contain in their genomic DNA a TTPA gene encoding an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 24, 49 or 37, or 25 (correctly SEQ ID NO: 57), 50, or 38, 54 or 59, 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 base sequence shown in SEQ ID NO: 26, 39, or 27 (correctly SEQ ID NO: 58), 40, or 55, and base sequences in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 26, 39, or 27 (correctly SEQ ID NO: 58), 40, or 55, or base sequences having 90% or more sequence identity. Furthermore, specific examples of genomic DNA of a phage containing such a gene include genomic DNA consisting of the base sequence shown in any one of SEQ ID NOs: 28, 29, 41, or 56, and a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in any one of SEQ ID NOs: 28, 29, 41, or 56; a base sequence having 90% or more sequence identity; a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 24, 37, or 25 (correctly SEQ ID NO: 57), 50, 38, 54, or 59; or a genomic DNA consisting of a base sequence having 80% or more sequence identity with a base sequence other than the base sequence encoding the amino acid sequence shown in SEQ ID NO: 24, 37, or 25 (correctly SEQ ID NO: 57), 50, 38, 54, or 59.

[0155] The sequence identity herein is not particularly limited. Specifically, for example, the sequence identity with respect to the reference sequence is 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% or more. or greater, 95.0% or greater, 95.5% or greater, 96.0% or greater, 96.5% or greater, 97.0% or greater, 97.5% or greater, 98.0% or greater, 98.5% or greater, 99.0% or greater, 99.1% or greater, 99.2% or greater, 99.3% or greater, 99.4% or greater, 99.5% or greater, 99.6% or greater, 99.7% or greater, 99.8% or greater, or 99.9% or greater sequence identity.

[0156] (2) Agriculturally acceptable carriers and media The term "agriculturally acceptable carriers and / or media" refers to substances that facilitate the application of the composition, can maintain the viability and infectivity of the phage, which is the active ingredient, and / or can 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 outdoors, and that are no or very little harmful to animals, especially humans.

[0157] (2-1) Carrier Specific examples of agriculturally acceptable carriers include surfactants, protective agents, and excipients. If desired, small amounts of wetting agents, emulsifiers, pH buffers, and the like may also be used. The carrier may be mixed in advance or may be mixed just before application.

[0158] Surfactants have the effect of improving the physicochemical properties of compositions on plants, such as wetting, emulsifying, dispersing, penetrating, adhesive, defoaming, and spreading properties. Surfactants can be used as the main component of agricultural chemical adjuvants called wetting agents. Examples of wetting agents include nonionic surfactants, combinations of nonionic and anionic surfactants, paraffin-based surfactants, and polyoxyethylene resin acid esters. More specific examples include polyoxyethylene alkyl ether compounds, polyoxyethylene fatty acid ester compounds, lignin sulfonate compounds, naphthylmethanesulfonate compounds, alkyl sulfosuccinate compounds, and tetraalkylammonium salt compounds.

[0159] In the case of phages, the protective agent is expected to have the effect of reducing damage caused by ultraviolet rays, etc. Examples of the protective agent include skim milk, casein, gelatin, etc.

[0160] Examples of excipients include glucose, lactose, sucrose, gelatin, starch, malt, and wheat flour.

[0161] (2-2) Solvent Specific examples of agriculturally acceptable solvents include water (including aqueous solutions), buffers, and liquid media. The solvent is preferably a sterile liquid.

[0162] (3) Other Active Ingredients In addition to the lytic agent described in the first aspect, the composition of the present invention may contain one or more other active ingredients having the same and / or different pharmacological action as long as they do not affect the lytic activity of the phage constituting the lytic agent.

[0163] The type of other active ingredient is not limited. For example, it may be a phage having lytic activity against the same and / or different bacteria. Examples of the phage having lytic activity against the same bacteria include other phages that specifically recognize and bind to Xanthomonas bacteria, similar to the phage constituting the lytic agent described in the first aspect.

[0164] Other active ingredients may include insecticides, herbicides, fertilizers (e.g., urea, ammonium nitrate, superphosphate), and, if necessary, known chemical pesticides, antibiotics, and biological pesticides.

[0165] 2-2-2. Dosage Form When used as a plant disease control composition, the composition of the present invention may be in any dosage form as long as it can maintain the infection site of the target bacterium on the target plant, the ability to colonize the target plant, and / or the ease with which the phage, the active ingredient, can infect the target bacterium. For example, the plant disease control composition may be suspended in an appropriate solution in the form of a liquid or wettable powder, or may be mixed with a carrier and solidified into a solid powder, granule, or gel. For example, when the infection site of the target bacterium on the target plant is the leaves, flowers, fruits, stems, branches, or trunk above ground, a liquid, wettable powder, or gel formulation is preferred, as these formulations are widely distributed over the infection site and have high colonization properties. On the other hand, when the infection site of the target bacterium is the roots or rhizomes below ground, a powder or granule formulation is preferred, as they can be slowly released in the soil and exert a sustained effect on the infection site.

[0166] 2-3. Application Method When the composition of the present invention is used as a plant disease control composition, any method known in the art may be used as long as it can apply the plant disease control composition to a target plant, and is not particularly limited. Since the phage, which is the active ingredient of the plant disease control composition, can invade the entire plant surface, such as the stems, leaves, and roots of a target plant, it can be applied by a method appropriate for the purpose. For example, if the application site is an above-ground part such as the stems and leaves, the plant disease control composition can be applied so that it directly contacts the application site. Direct contact can be achieved by, for example, painting, spraying, dusting, or immersion of the plant disease control composition on the application site. Application is particularly preferably carried out to a site of the target plant that is infected or at risk of infection by the target bacterium. Furthermore, if the application site is an underground part such as the roots, the composition can be applied indirectly by adding it to the soil, or to a medium if the application site is a culture medium. The term "soil" as used herein 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, the term "culture medium" refers to an artificially prepared culture medium for planting the target plant. It may be a solid medium such as an agar medium, or a liquid medium. Examples of culture medium include isolation beds, root-restricted pots, and seedbeds. The composition of the culture medium may be any known medium composition in the art. It can be selected appropriately depending on the type of plant, etc.

[0167] 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 bacterium of the present invention. They may be either angiosperms or gymnosperms. Furthermore, they may be herbaceous or woody plants. Suitable examples of target plants include agriculturally important plants, such as crop plants such as cereals, vegetables, and fruits, and ornamental plants. Specific examples of monocotyledonous plants include plants from the Poaceae family (e.g., rice, wheat, barley, corn, sugarcane, sorghum, sorghum, and turfgrass), plants from the Musaceae family (e.g., bananas), plants from the Amaryllidaceae family (e.g., leeks, onions, garlic, and chives), and plants from the Liliaceae family (e.g., lilies and tulips). In addition, dicotyledonous plants include Brassicaceae plants (e.g., cabbage, radish, Chinese cabbage, rapeseed), Asteraceae plants (e.g., lettuce, burdock, chrysanthemum), Fabaceae plants (e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, licorice), Solanaceae plants (e.g., tomato, eggplant, potato, tobacco, bell pepper, capsicum, petunia), and Rosaceae plants (e.g., strawberry, apple, pear, peach, loquat, almond, plum, rose, plum). , cherry blossom), Cucurbitaceae plants (e.g., cucumber, gourd, pumpkin, melon, watermelon), Anacardiaceae plants (e.g., mango, pistachio, cashew nut), Lauraceae plants (e.g., avocado), Rutaceae plants (e.g., mandarin orange, grapefruit, lemon, yuzu), Convolvulaceae plants (e.g., sweet potato), Theaceae plants (e.g., tea plant), and Vitaceae plants (e.g., grape).

[0168] 2-5. Target Plant Diseases The target plant diseases to which the plant disease control composition of the present invention is applied include any plant disease caused by the target bacterium of the present invention. Preferably, the plant disease is caused by a bacterium of the genus Xanthomonas. Examples include bacterial spot found on peaches, bacterial blight found on walnuts, bacterial pustule found on soybeans, angular leaf spot found on strawberries, bacterial blight found on tomatoes, bell peppers, lettuce, black rot found on cabbage, Chinese cabbage, broccoli, bacterial canker found on oranges and grapefruit, angular leaf spot found on cotton, and bacterial leaf blight found on rice.

[0169] 3. Plant Disease Control Method 3-1. Overview A third aspect of the present invention is a plant disease control method. The plant disease control method of the present invention is characterized by controlling plant diseases in a target plant by applying the plant disease control composition described in the second aspect to the target plant.

[0170] According to the method for controlling plant diseases of the present invention, plant diseases caused by bacteria, particularly bacteria of the genus Xanthomonas, can be controlled in target plants.

[0171] 3-2. Method The plant disease control method of the present invention includes a contact step as an essential step.

[0172] The "contact step" is a step of contacting a target plant with the plant disease control composition according to the second aspect. This step basically conforms to "2-3. Application method" for the plant disease control composition according to the second aspect.

[0173] In this aspect, "contact" refers to contact between the plant disease control composition and the target plant. More specifically, it refers to contact of the lytic agent according to the first aspect, i.e., the phage, which is the active ingredient of the plant disease control composition, with the plant body of the target plant, preferably with a site infected or at risk of infection by the target bacterium. The purpose of this step is to infect the target bacterium with the phage, which is the active ingredient, thereby lysing the target bacterium. As a result, the effect of controlling plant diseases caused by the target bacterium can be exerted.

[0174] Contact may be either direct or indirect. In this embodiment, direct contact refers to direct contact of the plant disease control composition with a predetermined site of the target plant. Specifically, for example, this refers to applying, spraying, dusting, or immersing the liquid or gel plant disease control composition on 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, indirect contact in this embodiment refers to contact of the plant disease control composition with a predetermined site of the target plant via an intermediary. For example, this refers to applying a granular plant disease control composition to the soil around the roots of the target plant. The phage, which is the active ingredient, is transported via water in the soil and eventually absorbed by the roots.

[0175] 3-3. Effects. Phages, the active ingredient of the composition obtained by the present invention, can efficiently kill target bacteria, thereby helping to prevent and suppress diseases caused by target bacteria. Furthermore, their specific bacteriolytic activity allows for the detection and identification of target bacteria, enabling disease diagnosis. While copper compounds and antibiotics have traditionally been used to treat Xanthomonas bacteria, these compounds can cause phytotoxicity and disrupt the balance of the microbiota. For example, some strains of Pseudomonas fluorescens have been demonstrated to promote plant growth (Haas D., Defago G., Nature Reviews in Microbiology, 2005, 3(4), 307-19). Therefore, copper compounds and antibiotics are likely to kill bacteria that are symbiotic with plants. However, because phages are derived from biological sources, no phytotoxicity has been reported, and their high specificity limits their impact on the balance of the microbiota.

[0176] 4. Method for Identifying Xanthomonas Bacteria 4-1. Overview A 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.

[0177] According to the present invention, it is possible to determine whether or not an unidentified plant pathogenic bacterium that has caused a plant disease is a bacterium of the genus Xanthomonas, and to identify it.

[0178] The identification method of the present invention includes a culturing step, a mixing step, a mixture culturing step, and a determination step as essential steps, and an isolation step as a selection step. Each step will be explained below.

[0179] (1) Isolation Step The "isolation step" is a step of isolating a test bacterium from plant tissue infected with a plant disease. This step is a selection step and may be performed as needed.

[0180] The term "test bacterium" refers to a plant pathogenic bacterium whose species has not been identified and which is subjected to the Xanthomonas bacterium identification method of the fourth aspect of the present invention or the Xanthomonas bacterium detection method of the fifth aspect described below.

[0181] The plant tissue may be any part of the plant affected by the plant disease, but preferably a part where the symptoms of the plant disease are clearly visible. For example, in the case of a peach affected by bacterial peach hole, the affected leaves may be used.

[0182] To isolate the test bacterium from harvested plant tissue, the diseased specimen may be immersed in a solvent such as water and extracted, optionally fragmented or crushed during extraction, and the extract may then be streaked onto an agar medium to pick a single colony.

[0183] (2) Culturing Step The "culturing step" is a step of culturing the isolated test bacterium to obtain a culture. The test bacterium may be cultured by a method known in the art.

[0184] The term "culture" refers to a substance obtained by culturing a test bacterium, and may be either liquid or solid.

[0185] Because the test bacterium in this step is unidentified, it is desirable to use a medium capable of culturing a wide range of plant pathogenic bacteria. A medium capable of culturing at least the Xanthomonas bacteria, the bacterium to be identified in this invention, is used. Such a medium may contain one or more components selected from the group consisting of proteolytic 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 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 peptone (sucrose, glutamic acid, peptone).

[0186] The isolated test bacterium is inoculated into the medium and cultured under appropriate culture conditions. Culture conditions include, for example, 20-40°C, 20-30°C, 22-28°C, or 24-26°C, with stirring to obtain a culture. The culture time is not limited, but may be, for example, until the turbidity at 600 nm reaches approximately 1.0. This step yields a culture solution of the test bacterium. The culture may also be a multi-stage culture consisting of two or more stages. For example, a soft agar-containing liquid medium may be added to the culture solution obtained after culture in a liquid medium, and the culture solution may be poured onto a solid medium such as an agar medium to solidify, after which further culture may be performed.

[0187] (3) Mixing Step The "mixing step" is a step of mixing the culture obtained in the culturing step with the lytic agent according to the first aspect to obtain a mixture.

[0188] The "mixture" refers to a mixture of a culture and a lysis agent, and may be either liquid or solid.

[0189] The method of mixing the culture and the lytic agent is not particularly limited as long as the lytic agent can be mixed. The lytic agent described in the first aspect may be administered in a solid state or in a liquid state suspended in water or a liquid medium.

[0190] If the culture and lysing agent are both liquid, the volume ratio of culture to lysing agent may 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 lysing agent may be thoroughly mixed by stirring or other means. On the other hand, when a soft agar-containing liquid medium is layered as described above, the culture is solid. In this case, the lysing agent may be dropped onto a solid culture such as a gel surface, allowing the two to be mixed on the solid medium to obtain a mixture.

[0191] (4) Mixture Cultivation Step The "mixture cultivation step" is a step of culturing the mixture under predetermined conditions.

[0192] When culturing the mixture, a soft agar-containing liquid medium may be added to the mixture, poured onto a solid medium such as an agar medium, and allowed to solidify, followed by further culturing.

[0193] The basic procedure of this step is similar to that of the culturing step described above. In this step, although not limited thereto, it is preferable to carry out culturing based on the so-called plaque assay method so as to easily confirm whether or not the test bacterium has been lysed by the phage constituting the lytic agent in the determination step described below. For example, a portion of the mixture may be mixed with a soft agar medium of the same composition, and then, before the soft agar medium solidifies, the mixture may be poured onto an agar medium of the same composition and spread over the entire medium. The mixture may then be cultured under the same conditions as in the culturing step described above.

[0194] (5) Determination Step The "determination step" is a step of determining that the test bacterium is a bacterium of the genus Xanthomonas when the test bacterium is lysed after the culture step.

[0195] The determination of the presence or absence of bacteriolysis is not limited, but for example, in the case of a plaque assay, the determination can be made based on the presence or absence of plaque formation. If plaques are present on the solidified soft agar medium after the aforementioned mixture culture step, this indicates that the test bacterium has been lysed by infection with the phage constituting the bacteriolytic agent of the present invention. Therefore, the test bacterium can be determined to be a bacterium of the genus Xanthomonas. On the other hand, if the test bacterium grows entirely on the agar medium and no plaques are present at all, the test bacterium can be determined not to be a bacterium of the genus Xanthomonas.

[0196] For more accurate determination, a negative control may be prepared by mixing with a medium containing no lytic agent in the mixture culture process, and / or a positive control may be prepared by using identified Xanthomonas bacteria in the culture process instead of the test bacteria, and it may be confirmed that no plaques form in the negative control and that plaques are observed in the positive control.

[0197] 4-3. Effects According to the method for identifying Xanthomonas bacteria of the present invention, it is possible to identify whether or not a plant disease is caused by Xanthomonas bacteria.

[0198] Furthermore, the method for identifying Xanthomonas bacteria of the present invention makes it possible to detect whether or not Xanthomonas bacteria are present in the lesions of a plant that has developed a plant disease that is suspected to be caused by Xanthomonas bacteria.

[0199] The examples in this application correspond to the examples in Japanese Patent Application No. 2022-156882 as follows: Example 1 corresponds to Example 1, Example 2 corresponds to Example 2, Example 3 corresponds to Example 3, Example 4 corresponds to Example 4, Example 5 corresponds to Example 5, Example 6 corresponds to Example 6, Example 7 corresponds to Example 7, Example 8 corresponds to Example 8, Example 9 corresponds to Example 9, and Example 10 corresponds to Example 10, respectively. Tables 1 to 13 in this application are completely identical to Tables 1 to 13 in Japanese Patent Application No. 2022-156882. Figures 1 to 11 in this application are completely identical to Figures 1 to 11 in Japanese Patent Application No. 2022-156882. SEQ ID NOs: 1 to 44 in this application are completely identical to SEQ ID NOs: 1 to 44 in Japanese Patent Application No. 2022-156882.

[0200] Example 1: Isolation of a novel bacteriophage and its lytic activity (1) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0201] (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). The bacteria used in this example are listed in Table 2 along with their NARO deposit number.

[0202]

[0203] For control purposes, a strain of environmentally beneficial Pseudomonas fluorescens, which has been reported to have plant growth promoting effects, was obtained from NCIMB, a research institute within the UK National Culture Collection (UKNCC) (NCIMB-ID: 10460).

[0204] For the cultivation of Xanthomonas oryzae pv. oryzae, a liquid medium (SW+P Broth) was used, prepared by dissolving 0.5 g of sodium L-glutamate, 1.0 g of MgCl₂·6H₂O, 0.1 g of KH₂PO₄, 5 g of sucrose, and 5 g of peptone in 1 L of H₂O and autoclaving. For the cultivation of other Xanthomonas species and Pseudomonas fluorescens, 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. Furthermore, agar medium was used by adding 15 g of agar per L to the above broth (SW+P Broth or YPG Broth) and autoclaving. (When SW+P Broth was used, this is referred to as "SW+P Agar," and when YPG Broth was used, this is referred to as "YPG Agar.") Furthermore, the soft agar medium (Top Agar) to be layered on top of the agar medium was prepared by adding 5 g of agarose per liter to the above broth, and the autoclaved Top Agar was stored at about 50°C and used as needed.

[0205] Each of the above strains, delivered in dry powder form, was suspended in 0.1 mL of broth and then streaked on agar (SW+P agar or YPG agar) at 25°C to isolate single colonies. The isolated colonies were inoculated into broth and cultured with shaking at 25°C to prepare the preculture medium. For the main culture, the preculture 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 cultivation was used as is as the bacterial suspension.

[0206] (2) Isolation and Purification of the First Phage. The novel phage was isolated from natural wastewater or soil obtained in Japan. The phage was isolated using a conventional 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. Equal amounts of the bacterial solution and the phage-containing solution were then mixed and left at room temperature for approximately 10 minutes. Next, 0.2 mL of the bacteria / phage mixture was added to 3 mL of Top Agar, quickly mixed using a vortex mixer, and poured onto the agar. After the Top Agar solidified, the mixture was incubated at 25°C for approximately 12 hours. Lytic plaques were formed on the bacterial lawn formed by the incubation. The gel from the plaques was then aspirated using a cut tip, and phages with lytic activity against Xanthomonas bacteria were isolated. Thereafter, the phage-containing liquid containing the isolated phages at a high concentration was used instead of the wastewater, and the procedure was repeated to purify the phages.

[0207] Using a method for detecting lytic plaques formed on soft agar media in which any of the above strains had been amplified, a total of three new phages were isolated from natural wastewater and soil. The three new phages isolated in Example 1 are referred to as "first phages."

[0208] The isolated phages were suspended in SM Buffer and passed through a 0.2 μm filter to recover the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the same conditions as above, and the phages were isolated again. This procedure was repeated several times to further purify the phages. The composition of SM Buffer is shown in Table 3.

[0209]

[0210] (3) Amplification and Purification of the First Phage To amplify and purify the isolated and purified first phage, a plate lysate (PL) method, an amplification method using a plaque assay, was performed. A bacteria / phage mixture was prepared so that many plaques would form on the agar, and then mixed with Top Agar. The mixture was then spread on YPG Agar and cultured. After that, 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 approximately 30 minutes. The supernatant was then passed through a 0.2 μm filter to recover the phage-containing solution.

[0211] The first phage was purified by adding 1 g of PEG 6000 (final concentration: 10%) and 0.4 g of NaCl (final concentration: 4%) to 10 mL of the recovered solution and dissolving the solution. The solution was then rotated overnight at 4°C using a rotator. The solution was then centrifuged at 15,000 x g at 4°C for 60 minutes, and the supernatant was removed. The recovered pellet was resuspended in 0.5 mL of SM Buffer. Next, 0.5 mL of chloroform was added, the solution was vigorously mixed, and the solution was left on ice for 6 hours. After centrifugation at 8,000 x g at 4°C for 10 minutes, the upper layer was carefully collected to obtain the purified phage solution. The concentration of the purified phage solution is generally expressed as a titer (PFU / mL) based on the number of plaques (Plaque Forming Units, PFU) in a plaque assay, and this titer serves as an index of bacteriolytic activity. The titer of the prepared first phage purified solution was determined by plaque assay using an appropriately diluted solution. 8 It was confirmed that the PFU / mL or more.

[0212] (4) Evaluation of the Host Range of the First Phage The host range of the first phage was evaluated using a spot test. 0.1 mL of the bacterial suspension alone was added to 3 mL of Top Agar, mixed, and poured onto the agar, allowing it to spread over the entire plate and solidify. In addition to the bacterial suspensions of each Xanthomonas bacterium listed in Table 1 prepared in (1) above, a control bacterial suspension of Pseudomonas fluorescens was also prepared. Approximately 5 μL of the purified phage solution was then added dropwise and incubated at 25°C for approximately 12 hours. If a clear circle (approximately 1 cm in diameter) formed at the drop location on the plate where the bacterial lawn had formed, the added phage was determined to have lytic activity against that bacterial strain.

[0213] An example of the results is shown in Figure 1. If the first phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The three types of first phages obtained by the present invention exhibited lytic activity against all strains belonging to the five bacterial species shown in Table 2: Xanthomonas arboricola, Xanthomonas campestris, Xanthomonas citri, Xanthomonas oryzae, and Xanthomonas cucurbitae. On the other hand, it was also confirmed that they did not exhibit lytic activity against Pseudomonas fluorescens. Although there have been reports of phages that exhibit activity against two or more species of Xanthomonas, the above-mentioned pattern has not been reported (Nakayinga R. et al., BMC Microbiology, 2021, 21:291).

[0214] Figure 12 shows an example of a plate from a plaque assay performed using the first phage having the nucleotide sequence set forth in SEQ ID NO:8 and Xanthomonas oryzae pv. oryzae (MAFF No. 311019) as the bacterial species. The formation of numerous plaques as a result of lysis indicates that the first phage also exhibits lytic activity against this bacterium isolated from rice. These results suggest that the first phage may be applicable to various plant diseases. For example, this suggests that the first phage may be useful in controlling peach bacterial hole, rice bacterial leaf blight, citrus canker, and broccoli black rot, which are caused by Xanthomonas bacteria, and is expected to be highly valuable for industrial use.

[0215] (5) Genome Analysis of the First Phage The genomic DNA sequence of the first phage was determined and analyzed.

[0216] (i) Preparation and sequencing of genomic DNA of the first phage TURBO DNA-free TMThe genome of the first phage was extracted using a kit (Thermo Fisher Scientific). Contaminating genomic DNA from the host bacterium was removed according to the kit's instructions. Subsequently, the phage coat molecules were degraded by Proteinase K treatment using a NucleoSpin® Virus (Machery-Nagel) according to the kit's instructions. After genomic DNA purification using a silica spin column, a genomic DNA solution of the first phage was prepared. The concentration of the genomic DNA was then measured using a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific), and 50 μL of genomic DNA solution was prepared to a final concentration of 0.2 ng / μL. Subsequently, the genome of the first phage was fragmented and adapter sequences were added by PCR using a Nextera XT DNA Library Prep (Illumina) according to the kit's instructions. Next, electrophoresis was performed using an Agilent High Sensitivity DNA Kit (Agilent Technologies) and a Bioanalyzer (Agilent Technologies). The average bp size of the samples was measured and the DNA fragment concentration was determined. Finally, measurement samples were prepared using a Miseq Reagent kit (Illumina) according to the accompanying manual, and measurements were performed using a Miseq next-generation sequencer (Illumina). The obtained data was preprocessed (e.g., trimmed) using CLC genomics workbench (Qiagen), and then de novo assembly was performed to obtain contig sequences corresponding to the phage genome sequence.

[0217] (ii) Bioinformatics analysis based on genome sequence information A BLAST analysis of the genome sequence (SEQ ID NOS: 8 to 10) of the first phage using GENETYX-NGS implemented in GENETYX revealed that the sequences of SEQ ID NOS: 8 to 10 share high sequence identity with each other. For example, the sequence identity (average nucleotide identity (ANI)) of the sequence of SEQ ID NOS: 8 to the sequence of SEQ ID NOS: 9 was 98% or higher over a range of 96% or higher of the entire length, and the sequence identity of the sequence of SEQ ID NOS: 8 to the sequence of SEQ ID NOS: 10 was 98% or higher over a range of 95% or higher of the entire length.

[0218] Furthermore, the tail fiber genes were searched for from the genome sequence information of the three phages. Searches for tail fiber genes were performed using the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ). The nucleotide sequences shown in SEQ ID NOS: 5 to 7, which are presumed to be tail fiber genes, were identified from the genome sequences of each phage. Analysis of the nucleotide sequences shown in SEQ ID NOS: 5 to 7 using GENETYX revealed that the nucleotide sequences of SEQ ID NOS: 5 to 7 share high sequence identity with each other. For example, the sequence identity of the nucleotide sequence of SEQ ID NOS: 5 to 6 and 7 was 96% or higher, and the sequence identity of the nucleotide sequences of SEQ ID NOS: 6 to 7 was 98% or higher. Furthermore, analysis of the amino acid sequences (SEQ ID NOS: 2 to 4) encoded by the nucleotide sequences shown in SEQ ID NOS: 5 to 7 using GENETYX revealed that the amino acid sequences of SEQ ID NOS: 2 to 4 also share high sequence identity with each other, with the sequence identity between the amino acid sequences of SEQ ID NOS: 2 to 4 being 98% or higher.

[0219] Based on the genome sequences of the above phages (SEQ ID NOS: 8-10), a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). As a result of the search, the genome sequence of phage Mija described in U.S. Patent Application Publication No. 2016 / 0309723 (SEQ ID NOS: 29) had the highest similarity score, with 86.5% sequence identity over 93% of the full length of the genome sequence of SEQ ID NOS: 8.

[0220] The lytic activity of phage Mija has been confirmed in Xylella fastidiosa, etc., and the host range of phage Mija differs from that of the phages of the present invention. In general, the tail fiber protein is thought to play a major role in the host specificity and host range characteristics of phages (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16:760-773). Therefore, the present inventors suspected that the difference in host range may be due to differences in the tail fiber gene.

[0221] Therefore, the identified nucleotide sequences of the tail fiber genes (SEQ ID NOS: 5 to 7) were used as queries to search for similar DNA sequences in the phage Mija genome sequence. As a result, only a region with 83% sequence identity over a range of only 16% of the entire length to the nucleotide sequence shown in SEQ ID NOS: 5 was found in the phage Mija genome sequence, and no DNA sequences homologous over the entire length to the nucleotide sequences shown in SEQ ID NOS: 5 to 7 were found. These results indicate that the differences in host range between phage Mija and the three phages obtained in the Examples of the present application are due to differences in the tail fiber genes.

[0222] To determine whether the tail fiber genes of the phages of the present invention are novel, a search for similar amino acid sequences was performed on the BLAST server provided by NCBI using the amino acid sequences of SEQ ID NOS: 2 to 4 as queries. As a result, the tail fiber protein of Stenotrophomonas phage vB_SmaS-DLP_6 (GenBank accession number: AMQ65898.1) had the highest similarity score. However, its amino acid sequence shared low sequence identity with any of the amino acid sequences of SEQ ID NOS: 2 to 4; for example, it shared only 58.47% sequence identity over only 35% of the full length with the sequence of SEQ ID NOS: 2. Note that Stenotrophomonas phage vB_SmaS-DLP_6 targets the genus Stenotrophomonas, a different genus of bacteria from the phages of the present invention.

[0223] The above results demonstrate that the phage of the present invention has a novel host range of the genus Xanthomonas that has not been reported previously, and that this characteristic is particularly mediated by the novel tail fiber gene.

[0224] Example 2: Isolation of a novel bacteriophage and its lytic activity (2) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0225] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in Example 2 are listed in Table 4 along with their respective NARO deposit number (MAFF No.).

[0226]

[0227] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (United Kingdom Microorganism Collection) (NCIMB-ID: 10460).

[0228] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0229] (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 in accordance with the methods described in "(2) Isolation and Purification of the First Phage" in Example 1. As a result, one new phage was isolated from the natural wastewater and soil. This novel phage isolated in Example 2 is referred to as the "second phage."

[0230] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0231] (3) Amplification and Purification of Second Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified second phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of First Phage" in Example 1. The titer of the prepared purified second phage solution was determined by plaque assay using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0232] (4) Evaluation of host range of second phage The host range of the second phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of first phage" in Example 1.

[0233] An example of the results is shown in Figure 2. If the second phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The second phage obtained by the present invention exhibited lytic activity against all of the Xanthomonas bacteria shown in Table 4. However, it did not exhibit lytic activity against Pseudomonas fluorescens. As in Example 1, no phage was previously known to exhibit lytic activity against all of the diverse bacterial species shown in Table 4.

[0234] Figure 13 shows an example of a plate from a plaque assay performed on this second phage using Xanthomonas oryzae pv. oryzae (MAFF No. 311019). Numerous plaques formed as a result of lysis, indicating that the second phage also exhibits lytic activity against this bacterium isolated from rice. These results suggest that the isolated second phage exhibits lytic activity against a wide range of Xanthomonas bacteria.

[0235] Furthermore, these results suggest that the second phage is useful for controlling diseases caused by Xanthomonas bacteria, such as peach bacterial hole, rice bacterial leaf blight, citrus canker, and broccoli black rot.

[0236] (5) Genome Analysis of the Second Phage The genomic DNA sequence of the second phage was determined and analyzed.

[0237] (i) Preparation and sequencing of the genomic DNA of the second phage TURBO DNA-free TM The genome of the second phage was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0238] (ii) Bioinformatics analysis based on genome sequence information Based on the genome base sequence (SEQ ID NO: 13) of the phage obtained in (i) above, a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI BLAST server (ibid.). As a result of the search, the genome sequence of a phage called Xp12 (access code: MT664984.1) targeting Xanthomonas bacteria had the highest similarity score, with a sequence identity of 98.42%. However, the only known bacterial species for which phage Xp12 exhibits bacteriolytic activity is Xanthomonas oryzae (Nakayinga R. et al., BMC Microbiology, 2021, 21:291).

[0239] To clarify the cause of the difference in host specificity between Xp12 and the second phage isolated in this example, the tail fiber protein sequences of both phage were compared. First, the tail fiber gene was identified from the genome sequence of the second phage in this example. The RAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ) were used to identify the gene. As a result, the nucleotide sequence shown in SEQ ID NO: 12 was identified as the tail fiber gene.

[0240] Furthermore, when the amino acid sequence (SEQ ID NO: 11) encoded by the gene consisting of the nucleotide sequence shown in SEQ ID NO: 12 was compared with the amino acid sequence of a protein related to Xp12 (access code: QNN97189.1), the types of amino acids at positions 278 and 350 were different. Specifically, valine at position 278 in Xp12 was changed to alanine, and serine at position 350 was changed to tryptophan. These are not conservative substitutions, and since serine in particular often contributes to protein-protein interactions, it is highly likely that these substitutions also result in differences in function.

[0241] From the above results, it was presumed that the second phage has a new host range of the genus Xanthomonas that had not been reported previously, and that this characteristic is mediated by the tail fiber gene.

[0242] Example 3: Isolation of a novel bacteriophage and its lytic activity (3) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0243] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). Each bacterium used in this example is listed in Table 5 along with its NARO deposit number (MAFF No.).

[0244]

[0245] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0246] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0247] (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 in accordance with the methods described in "(2) Isolation and purification of the first phage" in Example 1. As a result, one new phage was isolated from the natural wastewater and soil. This novel phage isolated in Example 3 is referred to as the "third phage."

[0248] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0249] (3) Amplification and Purification of the Third Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified third phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the prepared purified third phage solution was determined by plaque assay using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0250] (4) Evaluation of host range of the third phage The host range of the third phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0251] An example of the results is shown in Figure 3. If the third phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The third phage obtained by the present invention exhibited lytic activity against all Xanthomonas bacteria listed in Table 5. However, it did not exhibit lytic activity against Pseudomonas fluorescens. It is generally known that the host range of phages is limited to specific strains of specific local bacterial species (Sharma S. et al., Folia Microbiol., 2017, 62:17-55; Nakayinga R. et al., BMC Microbiology, 2021, 21:291). However, the phage isolated in this example exhibited lytic activity against all Xanthomonas bacteria isolated from different regions, as shown by the collection locations in Table 5. This suggests that the isolated third phage exhibits broad lytic activity against Xanthomonas bacteria.

[0252] Furthermore, these results suggest that the third phage may be useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0253] (5) Genome Analysis of the Third Phage The genomic DNA sequence of the third phage was determined and analyzed.

[0254] (i) Preparation and sequencing of genomic DNA of the third phage TURBO DNA-free TM The genome of the third phage was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0255] (ii) Bioinformatics Analysis Based on Genome Sequence Information: Based on the genome sequence (SEQ ID NO: 14) of the third phage obtained in (i) above, a search for similar DNA sequences and confirmation of sequence identity was performed using the BLAST server provided by NCBI (ibid.). The search results indicated that several Pseudomonas phage complete genomes had the highest similarity scores (access codes: KX129925.1, MN504636.1, KX898399.1, NC_041953.1, MW835180.1). However, all sequences shared only approximately 75% sequence identity within a small range of 6% to 7% of the total genome. Furthermore, the host bacteria of these phages are different from Xanthomonas bacteria. These results suggest that the third phage is a phage with a novel genome sequence with no known related genome sequences.

[0256] Example 4: Isolation of a novel bacteriophage and its lytic activity (4) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0257] (Materials and Methods) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). Each bacterium used in this example is listed in Table 6 along with its NARO deposit number (MAFF No.).

[0258]

[0259] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0260] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0261] (2) Isolation and Purification of the Fourth Phage The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of the First Phage" in Example 1. As a result, one new phage was isolated from the natural wastewater and soil. This novel phage isolated in Example 4 is referred to as the "fourth phage."

[0262] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0263] (3) Amplification and Purification of the Fourth Phage The isolated and purified fourth phage was amplified and purified by the plate lysate (PL) method, which is an amplification method using a plaque assay. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the prepared purified fourth phage solution was determined by the plaque assay method using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0264] (4) Evaluation of the host range of the fourth phage The host range of the fourth phage was evaluated by the spot test method. The basic procedure followed the method described in "(4) Evaluation of the host range of the first phage" in Example 1. An example of the results is shown in Figure 4. If the fourth phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The fourth phage obtained by the present invention exhibited lytic activity against all strains of Xanthomonas bacteria listed in Table 6. As in Example 3, it exhibited lytic activity against all Xanthomonas bacteria isolated from different regions. This suggests that the isolated phage exhibits broad lytic activity against Xanthomonas bacteria.

[0265] Furthermore, these results suggest that the fourth phage may be useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0266] (5) Genome analysis of the fourth phage The genomic DNA sequence of the fourth phage was determined and analyzed.

[0267] (i) Preparation and sequencing of the fourth phage genomic DNA TURBO DNA-free TM The phage genome was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of first phage (i) Preparation and sequencing of genomic DNA of first phage" in Example 1.

[0268] (ii) Bioinformatics Analysis Based on Genome Sequence Information Based on the genome sequence (SEQ ID NO: 17) of the phage obtained in (i) above, a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI BLAST server (ibid.). As a result of the search, several phages named FoX (accession codes: NC_055835.1, NC_055836.1, NC_055837.1, NC_055838.1) isolated in Belgium, as described in Nakayinga et al. (Nakayinga R. et al., BMC Microbiology, 2021, 21:291), had the highest similarity scores. However, with each sequence, there was only approximately 77% sequence identity over a range of approximately 10% of the total length. This result suggests that the phage isolated in Example 1 is essentially a phage with a novel genome sequence with no reported related sequences.

[0269] In general, tail fiber proteins are thought to play a major role in determining phage host specificity and host range (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16:760-773). Therefore, we searched for the tail fiber gene in the genome sequence of the fourth phage. Using the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ), we identified the nucleotide sequence shown in SEQ ID NO: 16, which is presumed to be the tail fiber gene, from the genome sequence of the fourth phage. This gene sequence shared 58% identity with the tail fiber gene of the known phage Fox. The reported host species of phage Fox is Xanthomonas campestris (Nakayinga R. et al., BMC Microbiology, 2021, 21:291). Therefore, its host range differs from that of the fourth phage.

[0270] Furthermore, these results suggest that it is difficult to predict the host range of phages carrying tail fiber genes with different nucleotide sequences based on the correlation between known tail fiber gene information and the host bacterial species information of the phages carrying those genes.

[0271] From the above results, it was presumed that the fourth phage has a new host range of the genus Xanthomonas that has not been reported previously, and that this characteristic is particularly mediated by the novel tail fiber gene.

[0272] Example 5: Isolation of a novel bacteriophage and its lytic activity (5) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0273] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). Each bacterium used in this example is listed in Table 7 along with its NARO deposit number (MAFF No.).

[0274]

[0275] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0276] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0277] (2) Isolation and Purification of the Fifth Phage The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of the First Phage" in Example 1. As a result, two new phages were isolated from the natural wastewater and soil. This novel phage isolated in Example 5 is referred to as the "fifth phage."

[0278] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0279] (3) Amplification and Purification of the Fifth Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified fifth phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the prepared purified fifth phage solution was determined by plaque assay using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0280] (4) Evaluation of host range of the fifth phage The host range of the fifth phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0281] An example of the results is shown in Figure 5. If the fifth phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The fifth phage obtained by the present invention exhibited lytic activity against all of the Xanthomonas bacteria listed in Table 7. However, it did not exhibit lytic activity against Pseudomonas fluorescens. As in Example 3, it exhibited lytic activity against all of the Xanthomonas bacteria isolated from different regions. This suggests that the isolated fifth phage exhibits broad lytic activity against Xanthomonas bacteria.

[0282] Furthermore, these results suggest that the fifth phage is useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0283] (5) Genome Analysis of the Fifth Phage The genomic DNA sequence of the fifth phage was determined and analyzed.

[0284] (i) Preparation and sequencing of genomic DNA of the fifth phage TURBO DNA-free TM The genome of the fifth phage was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0285] (ii) Bioinformatics Analysis Based on Genome Sequence Information Based on the genome base sequence of the fifth phage obtained in (i) (SEQ ID NOS: 18 and 19), a search for similar DNA sequences and confirmation of sequence identity was performed using the BLAST server provided by NCBI (ibid.). The search resulted in numerous hits of closely related sequences. The sequences with the highest sequence identity over 90% or more of the full length were Pseudomonas phage vB_PaeS (access code: LC552830.1) for SEQ ID NOS: 18 and P1940 (access code: MX298177.1) described in U.S. Patent Application Publication No. 2017-0319637 for SEQ ID NOS: 18 and 19, respectively. As shown in Table 8 below, each sequence showed similar sequence identity to both SEQ ID NOS: 18 and 19.

[0286]

[0287] In the table, the sequence identity is the numerical value for the range automatically aligned by the analysis server against the entire genome of SEQ ID NO: 18 or 19. The numerical value for that range is displayed as Query Cover. For example, the sequence identity of vB_PaeS, 98.28%, is calculated by limiting the region to 90% of the entire length of SEQ ID NO: 18. Therefore, although it is difficult to calculate the sequence identity for the entire length, it is estimated to be at least lower than 98.28%, which corresponds to a value lower than 90%. In the case of P1940, it is estimated to correspond to a value lower than 95%.

[0288] As a result of this analysis, it was revealed that the target bacteria of the phage having the genome of the above-mentioned known sequence are bacteria of the genus Pseudomonas. Therefore, although the fifth phage of this example has a very high sequence identity with the genome sequence of the known phage, it was difficult to identify it as that target bacterium. At the same time, phages that show a higher sequence identity to the fifth phage over a wider range than vB_PaeS or P1940 are likely to have a similar host range to the fifth phage of this example, i.e., bacteria of the genus Xanthomonas.

[0289] From the above results, it was presumed that the fifth phage has a new host range of the genus Xanthomonas, which had not been reported previously.

[0290] Example 6: Isolation of a novel bacteriophage and its lytic activity (6) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0291] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this study, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in this example are listed in Table 9 along with their respective NARO deposit number (MAFF No.).

[0292]

[0293] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0294] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0295] (2) Isolation and purification of the sixth phage The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and purification of the first phage" in Example 1. As a result, one new phage was isolated from the natural wastewater and soil. This novel phage isolated in Example 6 is referred to as the "sixth phage."

[0296] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0297] (3) Amplification and Purification of the Sixth Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified sixth phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the prepared purified sixth phage solution was determined by plaque assay using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0298] (4) Evaluation of host range of the sixth phage The host range of the sixth phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0299] An example of the results is shown in Figure 6. If the sixth phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The sixth phage obtained by the present invention exhibited lytic activity against all of the Xanthomonas bacteria listed in Table 9. However, it did not exhibit lytic activity against Pseudomonas fluorescens. As in Example 3, it exhibited lytic activity against all of the Xanthomonas bacteria isolated from different regions. This suggests that the isolated sixth phage exhibits broad lytic activity against Xanthomonas bacteria.

[0300] Furthermore, these results suggest that the sixth phage is useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0301] (5) Genome Analysis of the Sixth Phage The genomic DNA sequence of the sixth phage was determined and analyzed.

[0302] (i) Preparation and sequencing of genomic DNA of the sixth phage TURBO DNA-free TM The genome of the sixth phage was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0303] (ii) Bioinformatics Analysis Based on Genome Sequence Information: Based on the genome sequence (SEQ ID NO: 23) of the sixth phage obtained in (i) above, a search for similar DNA sequences and confirmation of sequence identity was performed using the BLAST server provided by NCBI (ibid.). As a result of the search, four phages (access codes: NC_017864.1, NC_007806.1, NC_029019.1, and NC_024381.1) showed 96.5-98.5% sequence identity over approximately 90% of the genome. The sequence identity over the entire length is estimated to be approximately 85%. However, despite this high sequence identity, the target bacteria of these known phages were not Xanthomonas but rather Pseudomonas or Stenotrophomonas. This suggests that the sixth phage is an entirely novel phage targeting Xanthomonas bacteria.

[0304] Therefore, to identify genes involved in the target bacterium's replication, we compared the genome sequences of the sixth phage with those of the four known phages. Furthermore, we identified ORFs in regions with low sequence identity to the known phages found through the comparison. The RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ) were used to identify ORFs.

[0305] The comparison revealed that the ranges of positions 8062 to 9473 and positions 31995 to 34230 in the genome sequence shown in SEQ ID NO: 23 were regions with low sequence identity to known phages. A total of five ORFs were detected in this region. Specific ORFs were DNA regions represented by the base sequences of positions 8082 to 8573, 8645 to 8842, 9223 to 9477, 31881 to 32789, and 32792 to 33694, respectively, in the genome sequence of SEQ ID NO: 23.

[0306] To clarify which genes are important for determining the host range, each ORF was examined. As a result, the most common hits were nucleotide sequences with 30% or more sequence identity with known bacteriophages targeting Xanthomonas bacteria. Therefore, in the sixth phage isolated in Example 6, the nucleotide sequence from positions 9223 to 9477 in the genome sequence shown in SEQ ID NO: 23 (SEQ ID NO: 22) showed high sequence identity and was presumed to be an important gene region.

[0307] To determine whether this gene is a known gene, a search was conducted using the BLAST server provided by NCBI based on the amino acid sequence of the translation product (SEQ ID NO: 21) to search for similar amino acid sequences not limited to bacteriophages targeting Xanthomonas bacteria. As a result, even the sequence with the highest similarity score had a sequence identity of only about 50%, and no sequences showing high sequence identity were detected.

[0308] Example 7: Isolation of a novel bacteriophage and its lytic activity (7) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0309] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this study, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in this example are listed in Table 10 along with their respective NARO deposit number (MAFF No.).

[0310]

[0311] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0312] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0313] (2) Isolation and Purification of the Seventh Phage The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of the First Phage" in Example 1. As a result, two new phages were isolated from the natural wastewater and soil. This novel phage isolated in Example 7 is referred to as the "seventh phage."

[0314] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0315] (3) Amplification and Purification of the Seventh Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified seventh phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the prepared seventh phage purified solution was determined by plaque assay using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0316] (4) Evaluation of host range of the seventh phage The host range of the seventh phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0317] An example of the results is shown in Figure 7. If the seventh phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The seventh phage obtained by the present invention exhibited lytic activity against all of the Xanthomonas bacteria listed in Table 10. However, it did not exhibit lytic activity against Pseudomonas fluorescens. As in Example 1, it exhibited lytic activity against a wide variety of bacterial species, and also exhibited lytic activity against all of the strains isolated in different regions. This suggests that the isolated seventh phage exhibits broad lytic activity against Xanthomonas bacteria.

[0318] Furthermore, these results suggest that the seventh phage is useful for controlling bacterial borer disease of peach and black rot disease of broccoli, which are caused by bacteria of the genus Xanthomonas.

[0319] (5) Genome Analysis of the Seventh Phage The genomic DNA sequence of the seventh phage was determined and analyzed.

[0320] (i) Preparation and sequencing of genomic DNA of the seventh phage TURBO DNA-free TM The genome of the seventh phage was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0321] (ii) Bioinformatics analysis based on genome sequence information Based on the genome sequence of the seventh phage obtained in (i) (SEQ ID NOs: 28 and 29), a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI BLAST server (ibid.). As a result of the search, the genome sequence of the phage described in JP 2021-102635 A (SEQ ID NOs: 19 and 20 in the document (SEQ ID NOs: 30 and 31 in the present specification, respectively)) had the highest similarity score. Both sequences shared approximately 92% sequence identity over approximately 90% of their full length. From these results, it is estimated that the sequence identity over the entire length is approximately 85%. Furthermore, the genome sequence of ΦXc10 (Nakayinga R. et al., BMC Microbiology, 2021, 21:291) had the next highest similarity score. The genome sequence of ΦXc10 (access code: NC_047840.1) shared approximately 92% sequence identity over 87% of its length. Based on these results, the overall sequence identity is estimated to be approximately 80%. Furthermore, the genome sequences of f30-Xaj and f20-Xaj (Nakayinga R. et al., 2021, supra) also had similarly high similarity scores. The genome sequences of f30-Xaj and f20-Xaj (access codes: NC_030937.1 and NC_030928.1) shared approximately 91% sequence identity over 74% of their length. Based on these results, the overall sequence identity is estimated to be approximately 70%. However, none of these phages targeted Xanthomonas arboricola pv. pruni.

[0322] To investigate the cause of the difference in target bacteria, a search for novel genes was conducted in the genome sequence information of the seventh phage (SEQ ID NO: 28). The RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ) were used to predict the gene regions. As a result of the search, a group of genes encoding novel tail tubular proteins was identified as genes involved in the host range of the seventh phage. These gene regions were located in regions with identical base sequences in the genome sequences of the two types of seventh phage obtained in (i) above.

[0323] SEQ ID NO: 26 shows the nucleotide sequence of the tailtube protein A gene contained in the genome sequence (SEQ ID NO: 28) of the seventh phage discovered in this example, and SEQ ID NO: 24 shows the amino acid sequence of tailtube protein A. Meanwhile, SEQ ID NO: 27 (correctly SEQ ID NO: 58) shows the nucleotide sequence of the tailtube protein B gene, and SEQ ID NO: 25 (correctly SEQ ID NO: 57) shows the amino acid sequence of tailtube protein B.

[0324] Tailtube protein A and tailtube protein B have been reported to be involved in specific adsorption of phages to bacteria (Maozhi Hu, et al., 2020, 9:1, 855-867), suggesting that they are likely involved in the host range of the seventh phage. However, when a search was performed on the NCBI BLAST server based on the amino acid sequences of tailtube protein A (SEQ ID NO: 24) and tailtube protein B (SEQ ID NO: 25 (correctly SEQ ID NO: 57)), no amino acid sequences shared 97% or more sequence identity. The highest sequence identity, approximately 96%, was with the amino acid sequences of the tailtube proteins of the aforementioned ΦXc10 and f20-Xaj.

[0325] These findings suggest that these proteins are novel tail-tube proteins that mediate the lytic activity against Xanthomonas bacteria.

[0326] Example 8: Isolation of a novel bacteriophage and its lytic activity (8) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0327] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this study, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in this example are listed in Table 11 along with their respective NARO deposit number (MAFF No.).

[0328]

[0329] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0330] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0331] (2) Isolation and Purification of the Eighth Phage The novel phage was isolated from natural wastewater, soil, or plant bodies (leaves) obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of the First Phage" in Example 1. As a result, five new phages were isolated from natural wastewater, soil, or water in which plant bodies had been soaked. This novel phage isolated in Example 8 is referred to as the "eighth phage."

[0332] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0333] (3) Amplification and Purification of the Eighth Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified eighth phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the prepared eighth phage purified solution was determined by plaque assay using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0334] (4) Evaluation of host range of the eighth phage The host range of the eighth phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0335] Examples of the results are shown in Figures 8 and 9. If the eighth phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The eighth phage obtained by the present invention exhibited lytic activity against all of the Xanthomonas bacteria listed in Table 11. However, it did not exhibit lytic activity against Pseudomonas fluorescens. As in Example 3, it exhibited lytic activity against all of the Xanthomonas bacteria isolated from different regions. This suggests that the isolated eighth phage exhibits broad lytic activity against Xanthomonas bacteria.

[0336] Furthermore, these results suggest that the eighth phage is useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0337] (5) Genome analysis of the eighth phage The genomic DNA sequence of the eighth phage was determined and analyzed.

[0338] (i) Preparation and sequencing of genomic DNA of the eighth phage TURBO DNA-free TM The genome of the eighth phage was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0339] (ii) Bioinformatics analysis based on genome sequence information Based on the genome sequence of the eighth phage obtained in (i) (SEQ ID NOS: 18 and 19), a search for similar DNA sequences and confirmation of sequence identity were performed using the BLAST server provided by NCBI (ibid.). As a result of the search, no sequences sharing 80% or more sequence identity over 50% or more of the entire length with any of the sequences of SEQ ID NOS: 32 to 36 were found. Furthermore, no sequences sharing 90% or more sequence identity over 20% or more of the entire length with any of the sequences of SEQ ID NOS: 32 to 36 were found. These results suggest that the eighth phages isolated in Example 8 are essentially phages with novel genome sequences with no reported related sequences.

[0340] Furthermore, the sequence identity between the genomic DNA sequences of each phage of SEQ ID NOs: 32 to 36 was analyzed by BLAST analysis using GENETYX-NGS implemented in the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ). The sequence identity, measured as the average nucleotide identity (ANI), is shown in Table 12 below.

[0341]

[0342] In Table 12, the ANI value and comparison range of the genomic DNA sequence in each row relative to the genomic DNA sequence in each column are shown in the format of "ANI value (%) / comparison range (%)." Note that the comparison range (%) in Table 12 is the percentage of the region in the genomic DNA sequence in the corresponding column that is aligned with the genomic DNA sequence in the corresponding row. For example, if the ANI value (%) / comparison range (%) is "90 / 90," the genomic DNA sequence in the corresponding row has 90% sequence identity with the genomic DNA sequence in the corresponding column over 90% of its entire length.

[0343] Because the genomic DNA sequences of the phages represented by SEQ ID NOs: 32 to 36 have different lengths, the ANI values ​​and comparison ranges for the genomic DNA sequences in each column of the genomic DNA sequences in each row in Table 12 may be slightly different from the ANI values ​​and comparison ranges when the rows and columns are interchanged. For example, when the ANI values ​​and comparison ranges for the row of SEQ ID NO: 32 and the column of SEQ ID NO: 35 are compared with the ANI values ​​and comparison ranges for the row of SEQ ID NO: 35 and the column of SEQ ID NO: 32, the ANI values ​​are 100% in both cases, but because the genome lengths of SEQ ID NOs: 32 and 35 are different, the numerical values ​​of the comparison ranges are different.

[0344] As shown in Table 12, the base sequences of SEQ ID NOs: 32 to 36 were found to have 98 to 100% sequence identity with each other in the range of 90% to 100%, and were therefore very similar to each other. When the ANI values ​​and comparison ranges in Table 12 were multiplied to estimate the overall sequence identity, SEQ ID NO: 35 had the lowest overall sequence identity to SEQ ID NO: 36, at 90%.

[0345] Comparison of the genomic DNA sequences of the above phages showed that phages having a genomic DNA sequence containing a nucleotide sequence having 90% or more sequence identity with any of the nucleotide sequences of SEQ ID NOs: 32 to 36, i.e., phages having a genomic DNA sequence containing a nucleotide sequence in which approximately 10% of the nucleotides in any of the nucleotide sequences of SEQ ID NOs: 32 to 36 have added, deleted, and / or substituted nucleotides, also have essentially the same host range and lytic activity as phages having a genomic DNA sequence of any of SEQ ID NOs: 32 to 36.

[0346] Example 9: Isolation of a novel bacteriophage and its lytic activity (9) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0347] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this study, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in this example are listed in Table 13 along with their respective NARO deposit number (MAFF No.).

[0348]

[0349] For control purposes, a strain of environmentally beneficial Pseudomonas fluorescens, which has been reported to have plant growth promoting effects, was obtained from NCIMB, a research institute within the UK National Culture Collection (UKNCC) (NCIMB-ID: 10460).

[0350] Various Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0351] (2) Isolation and Purification of the Ninth Phage The novel phage was isolated from natural wastewater, soil, or plant bodies (leaves) obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of the First Phage" in Example 1. As a result, five new phages were isolated from natural wastewater, soil, or water in which plant bodies had been soaked. This novel phage isolated in Example 9 is referred to as the "ninth phage."

[0352] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0353] (3) Amplification and Purification of the Ninth Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified Ninth phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the prepared purified Ninth phage solution was determined by plaque assay using an appropriately diluted solution, and the titer was 10 8 It was confirmed that the PFU / mL or more.

[0354] (4) Evaluation of host range of phage No. 9 The host range of phage No. 9 was evaluated by spot testing, following the basic procedure described in "(4) Evaluation of host range of phage No. 1" in Example 1.

[0355] An example of the results is shown in Figure 10. When a phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped becomes transparent. The phage obtained by the present invention exhibited lytic activity against all strains belonging to the three bacterial species listed in Table 13: Xanthomonas arboricola, Xanthomonas citri, and Xanthomonas campestris. Furthermore, for Xanthomonas arboricola and Xanthomonas campestris, lytic activity was also observed against strains of different pathotypes. On the other hand, it was confirmed that no lytic activity was exhibited against Pseudomonas fluorescens. While there have been reports of phages active against two or more species of Xanthomonas, the above-mentioned pattern, including pathotypes, has not been reported (Nakayinga R. et al., BMC Microbiology, 2021, 21:291). This result demonstrates the potential application of the phage of the present invention to various plant diseases. For example, it has been suggested that the compound is useful for controlling bacterial peach hole, citrus canker, and broccoli black rot, which are caused by bacteria of the genus Xanthomonas, and is expected to have great value in industrial applications.

[0356] (5) Genome analysis of the ninth phage The genomic DNA sequence of the ninth phage was determined and analyzed.

[0357] (i) Preparation and sequencing of genomic DNA of the 9th phage TURBO DNA-free TM The genome of the ninth phage was extracted using a kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0358] (ii) Bioinformatics Analysis Based on Genome Sequence Information: Based on the genome sequence of the ninth phage (SEQ ID NO: 41) obtained in (i) above, a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI BLAST server (ibid.). The search results showed that the genome sequence of the phage "Xanthomonas phage Xaa_vB_phi31" (NCBI access code: MT951568.1) listed in the NCBI database had the highest similarity score. It shared approximately 85% sequence identity over approximately 80% of its entire length. Based on these results, the overall sequence identity is estimated to be approximately 68%. The only known host for Xaa_vB_phi31 was Xanthomonas euvesicatoria pv. allii XaaBL11. Regarding the other phages with high similarity scores, there was no information suggesting that they exhibited broad lytic activity against various species of Xanthomonas bacteria, as did the ninth phage.

[0359] To investigate the cause of the difference in target bacteria, we searched for novel genes in the genome sequence information of the ninth phage (SEQ ID NO: 41). The RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ) were used to predict the gene region. As a result of the search, a group of genes encoding novel tail tubular proteins was identified as genes involved in the phage host range.

[0360] SEQ ID NO: 39 shows the nucleotide sequence of the tailtube protein A gene contained in the genome sequence (SEQ ID NO: 41) of the ninth phage discovered in this example, and SEQ ID NO: 37 shows the amino acid sequence of tailtube protein A. Meanwhile, SEQ ID NO: 40 shows the nucleotide sequence of the tailtube protein B gene, and SEQ ID NO: 38 shows the amino acid sequence of tailtube protein B.

[0361] Tailtube protein A and tailtube protein B have been reported to be involved in specific phage adsorption to bacteria (Maozhi Hu, et al., 2020, 9:1, 855-867), suggesting their possible involvement in the host range of the ninth phage. However, when a search was performed on the NCBI BLAST server based on the amino acid sequences of tailtube protein A (SEQ ID NO: 37) and tailtube protein B (SEQ ID NO: 38), no amino acid sequences shared 97% or more sequence identity. The highest sequence identity for tailtube protein A was approximately 95% for the amino acid sequence of the Xanthomonas phage Xaa_vB_phi31. The sequence identity between the nucleotide sequence of SEQ ID NO: 39 and the nucleotide sequence encoding tailtube protein A of Xaa_vB_phi31 was approximately 89%. The amino acid sequence of tailtube protein B of Xaa_vB_phi31 also showed the highest sequence identity of approximately 91%, and the nucleotide sequence of the gene showed approximately 86% sequence identity with the nucleotide sequence of SEQ ID NO:40.

[0362] These findings suggest that these proteins are novel tail-tube proteins that mediate the lytic activity against Xanthomonas bacteria.

[0363] Example 10: Isolation of a novel bacteriophage and its lytic activity (10) (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0364] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). The bacteria used in this example are the same as those listed in Table 13 of Example 9.

[0365] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0366] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0367] (2) Isolation and Purification of the Tenth Phage The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of the First Phage" in Example 1. As a result, one new phage was isolated from the natural wastewater and soil. This novel phage isolated in Example 10 is referred to as the "tenth phage."

[0368] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0369] (3) Amplification and Purification of the Tenth Phage The plate lysate (PL) method was carried out to amplify and purify the isolated and purified phage. The specific method was in accordance with the method described in "(3) Amplification and Purification of the First Phage" in Example 1. The titer of the purified solution of the tenth phage was determined by plaque assay using an appropriately diluted solution. 8 It was confirmed that the PFU / mL or more.

[0370] (4) Evaluation of host range of the tenth phage The host range of the tenth phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0371] An example of the results is shown in Figure 11. If the phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The tenth phage exhibited lytic activity against all strains belonging to the three bacterial species listed in Table 13: Xanthomonas arboricola, Xanthomonas citri, and Xanthomonas campestris. Furthermore, for Xanthomonas arboricola and Xanthomonas campestris, it also exhibited lytic activity against strains of different pathotypes. On the other hand, it was confirmed that it did not exhibit lytic activity against Pseudomonas fluorescens. While there have been reports of phages active against two or more Xanthomonas species, the above-mentioned pattern, including pathotypes, has not been reported (Nakayinga R. et al., BMC Microbiology, 2021, 21:291). This result demonstrates the potential application of the tenth phage to various plant diseases. For example, it has been suggested that the compound is useful for controlling bacterial peach hole, citrus canker, and broccoli black rot, which are caused by bacteria of the genus Xanthomonas, and is expected to have great value in industrial applications.

[0372] (5) Genome analysis of the tenth phage The genomic DNA sequence of the tenth phage was determined and analyzed.

[0373] (i) Preparation and sequencing of genomic DNA of the tenth phage TURBO DNA-free TM The genome of the tenth phage was extracted using a kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0374] (ii) Bioinformatics analysis based on genome sequence information Based on the genome base sequence (SEQ ID NO: 44) of the tenth phage obtained in (i) above, a search for similar DNA sequences and confirmation of sequence identity was performed using the NCBI BLAST server (ibid.). As a result of the search, the phage isolated in the United States (name: RiverRider; NCBI access code: NC_048703.1) described in Miller et al. (Miller M. et al., Archives of Virology, 2020, 165:1481-1484) had the highest similarity score. It shared approximately 91% sequence identity with SEQ ID NO: 44 over approximately 80% of its full length, and the sequence identity over the entire genome sequence was less than 80% (equivalent to approximately 73%). These results indicated that the tenth phage isolated in this example is a phage with a novel genome sequence.

[0375] In general, tail fiber proteins are thought to play a major role in determining phage host specificity and host range (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16:760-773). Therefore, we searched for tail fiber genes in the genome sequence information of the phages. Using the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ), we identified the nucleotide sequence shown in SEQ ID NO: 43, which is a putative tail fiber gene, from the genome sequence of the tenth phage. A search for similar DNA sequences for this gene sequence revealed that the tail fiber gene sequence of the aforementioned phage RiverRider had the highest similarity score. The sequence identity of this nucleotide sequence was approximately 77% across the first half of the nucleotide sequence of SEQ ID NO: 43 (residues 1 to 890, corresponding to approximately 52% of the total length). The tail fiber protein of RiverRider also had the highest similarity score for the amino acid sequence of SEQ ID NO: 42. The amino acid sequence identity was approximately 82% over approximately 52% of the entire length, indicating that the tail fiber protein of the 10th phage is a novel protein with no closely related proteins, and is a particularly distinctive protein of the phage of the present invention compared with known phages.

[0376] Regarding the host bacteria of phage RiverRider, the above-mentioned literature states that it exhibited lytic activity against Xanthomonas fragariae but not against Xanthomonas arboricola or Xanthomonas campestris. Therefore, its host range is clearly different from that of the 10th phage, suggesting that the large difference in host range between this phage RiverRider and the phage obtained in this example is due to differences in the tail fiber genes.

[0377] From the above results, it was presumed that the 10th phage has a new host range of the genus Xanthomonas that has not been reported previously, and that this characteristic is particularly mediated by the novel tail fiber gene.

[0378] Example 11: Isolation of a novel second bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0379] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this example, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). In addition to the bacteria listed in Table 4, the bacteria listed in Table 14 were also used.

[0380]

[0381] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (United Kingdom Microorganism Collection) (NCIMB-ID: 10460).

[0382] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0383] (2) Isolation and Purification of Phages The novel phages were isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of First Phage" in Example 1. As a result, two new phages were isolated from the natural wastewater and soil.

[0384] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0385] (3) Amplification and Purification of Phages The plate lysate (PL) method was carried out to amplify and purify the isolated and purified phages. The specific method was in accordance with the method described in "(3) Amplification and Purification of First Phage" in Example 1. The titer of the prepared purified phage solution was determined by plaque assay using an appropriately diluted solution.8 It was confirmed that the PFU / mL or more.

[0386] (4) Evaluation of host range of phage The host range of the phage was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of first phage" in Example 1.

[0387] Examples of the results are shown in Figures 14 and 15. When a phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped becomes transparent. Like the second phage, the two phages obtained by the present invention exhibited lytic activity against all Xanthomonas bacteria listed in Table 4 (Figure 14). However, they did not exhibit lytic activity against Pseudomonas fluorescens. This suggests that the two isolated phages may be highly similar to the second phage. Furthermore, the lytic activity against Xanthomonas campestris pv. campestris was confirmed for the second phage obtained in Example 2 and the two phages obtained in this Example. As a result, all three phages exhibited lytic activity against this strain as well (Figure 15).

[0388] Furthermore, these results suggest that the phages obtained in this example are useful for controlling diseases caused by Xanthomonas bacteria, such as peach bacterial bore, rice bacterial leaf blight, citrus canker, and broccoli black rot.

[0389] (5) Genome Analysis of Phages The genomic DNA sequences of the phages obtained in this example were determined and analyzed.

[0390] (i) Preparation and sequencing of phage genomic DNA TURBO DNA-free TM The genomes of the two phages were extracted using a kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of the first phage (i) Preparation and sequencing of genomic DNA of the first phage" in Example 1.

[0391] (ii) Bioinformatics analysis based on genome sequence information Based on the genome sequences (SEQ ID NOs: 47 and 48) of the phages obtained in (i) above, the average nucleotide identity (ANI) relative to the genome sequence of the second phage (SEQ ID NO: 13) was analyzed using GENETYX-NGS. As a result of the analysis, the average nucleotide identity (ANI) relative to SEQ ID NO: 13 was 98.90% for SEQ ID NO: 47 and 98.92% for SEQ ID NO: 48. For this reason, the two phages obtained in this example, together with the phage obtained in Example 2, are referred to as the "second phage."

[0392] To clarify the cause of the identical host specificity between the phage isolated in this example and the phage isolated in Example 2, the sequences of the tail fiber proteins of both phages were compared.

[0393] First, the tail fiber genes were identified from the genome sequences of the two phages in this example. The RAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ) were used to identify the genes. As a result, the nucleotide sequence shown in SEQ ID NO: 46 was identified as the nucleotide sequence of the tail fiber gene. The nucleotide sequences of the tail fiber genes of the two phages isolated in this example were identical.

[0394] Furthermore, the amino acid sequence (SEQ ID NO: 45) encoded by the gene consisting of the nucleotide sequence shown in SEQ ID NO: 46 was compared with the amino acid sequence (SEQ ID NO: 11) encoded by the tail fiber gene of the phage isolated in Example 2. As a result, it was found that only the type of amino acid at position 154 was different. Specifically, threonine (Thr) at position 154 in SEQ ID NO: 11 had been changed to alanine (Ala).

[0395] In relation to phage Xp12, which had the highest similarity score to the phage isolated in Example 2, the amino acid sequence of its related protein differed in the types of amino acids at positions 278 and 350, as with the phage isolated in Example 2, and the phage obtained in this example also differed in the type of amino acid at position 154, as mentioned above.

[0396] As described above in Example 2, the only known bacterial species in which phage Xp12 exhibits lytic activity is Xanthomonas oryzae, suggesting that the lytic activity of the second phage against a wide range of hosts is due to differences in the amino acids at positions 278 and 350 in the amino acid sequence of the tail fiber protein. Furthermore, it was suggested that even a phage having a different amino acid at another additional position, such as the second phage obtained in this example, has a wide host range.

[0397] Example 12: Isolation of a novel seventh bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0398] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In the present invention, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). In addition to the Xanthomonas bacteria used in Example 7 shown in Table 10, the bacteria used in this example are listed in Table 15 along with their NARO deposit number (MAFF No.).

[0399]

[0400] For control purposes, a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UKNCC (NCIMB-ID: 10460).

[0401] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0402] (2) Isolation and Purification of Phages The novel phage was isolated from natural wastewater or soil obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of First Phage" in Example 1. As a result, one new phage was isolated from the natural wastewater and soil.

[0403] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0404] (3) Amplification and Purification of Phages The plate lysate (PL) method was carried out to amplify and purify the isolated and purified phages. The specific method was in accordance with the method described in "(3) Amplification and Purification of First Phage" in Example 1. The titer of the prepared purified phage solution was determined by plaque assay using an appropriately diluted solution. 8 It was confirmed that the PFU / mL or more.

[0405] (4) Evaluation of host range of phage The host range of the obtained phages was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of first phage" in Example 1.

[0406] Examples of the results are shown in Figures 16 and 17. When a phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped becomes transparent. The phages obtained by the present invention exhibited lytic activity against all of the Xanthomonas bacteria listed in Table 10 (Figure 16). However, they did not exhibit lytic activity against Pseudomonas fluorescens. This suggests that the isolated phages may be highly similar to the seventh phage. Furthermore, the two seventh phages obtained in Example 7 and the phages obtained in this Example were tested for lytic activity against Xanthomonas campestris pv. vesicatoria. As a result, all three phages exhibited lytic activity against this strain as well (Figure 17).

[0407] Furthermore, these results suggest that the phages obtained in this example are useful for controlling bacterial borer disease of peach and black rot disease of broccoli, which are caused by bacteria of the genus Xanthomonas.

[0408] (5) Genome Analysis of the Seventh Phage The genomic DNA sequence of the phage obtained in this example was determined and analyzed.

[0409] (i) Preparation and sequencing of phage genomic DNA TURBO DNA-free TM The phage genome was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of first phage (i) Preparation and sequencing of genomic DNA of first phage" in Example 1.

[0410] (ii) Bioinformatics Analysis Based on Genome Sequence Information Based on the genome sequence (SEQ ID NO: 53) of the phage obtained in (i) above, the average nucleotide identity (ANI) to the genome sequence of the seventh phage (SEQ ID NOs: 28 and 29) was analyzed using GENETYX-NGS. As a result, the sequence shown in SEQ ID NO: 53 had 92.61% sequence identity over a range of 75.06% with the sequence shown in SEQ ID NO: 28. On the other hand, the sequence shown in SEQ ID NO: 29 had 92.19% sequence identity over a range of 77.05%. It was unexpected that the identity of the genome sequences was not extremely high.

[0411] To clarify the cause of the identical host specificity between the phages isolated in this example and those isolated in Example 7, the sequences of the tailtube proteins of both phages were compared.

[0412] First, the gene cluster encoding the tailtube protein was identified from the genome sequence of the phage in this example. The BRAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ) were used to identify the genes.

[0413] SEQ ID NO: 51 shows the nucleotide sequence of the tailtube protein A gene contained in the genome sequence (SEQ ID NO: 53) of the phage discovered in this example, and SEQ ID NO: 49 shows the amino acid sequence of tailtube protein A. Meanwhile, SEQ ID NO: 52 shows the nucleotide sequence of the tailtube protein B gene, and SEQ ID NO: 50 shows the amino acid sequence of tailtube protein B.

[0414] Furthermore, the amino acid sequences of these tailtube proteins (SEQ ID NOs: 49 and 50) were compared with the amino acid sequences of the tailtube proteins of the phages isolated in Example 7 (SEQ ID NOs: 24 and 58). As a result, the sequence identity of the amino acid sequence of tailtube protein A was 96.58%, and the sequence identity of the amino acid sequence of tailtube protein B was 97.60%. For this reason, the phages obtained in this example, together with the phages obtained in Example 7, are referred to as the "seventh phages."

[0415] Specifically, with regard to the amino acid sequence of tailtube protein A, the amino acid sequence of SEQ ID NO: 49 showed the following seven amino acid substitutions from the amino acid sequence shown in SEQ ID NO: 24: glutamic acid (Glu) at position 28 replaced by aspartic acid (Asp), serine (Ser) at position 108 replaced by asparagine (Asn), glutamine (Gln) at position 110 replaced by histidine (His), glutamine (Gln) at position 153 replaced by lysine (Lys), aspartic acid (Asp) at position 157 replaced by asparagine (Asn), tyrosine (Tyr) at position 189 replaced by phenylalanine (Phe), and valine (Val) at position 201 replaced by tyrosine (Tyr).

[0416] On the other hand, with regard to the amino acid sequence of tailtube protein B, the amino acid sequence of SEQ ID NO:50 showed the following 20 amino acid substitutions from the amino acid sequence of SEQ ID NO:58: alanine (Ala) to proline (Pro) at position 25, alanine (Ala) to serine (Ser) at position 53, alanine (Ala) to proline (Pro) at position 216, histidine (His) to tyrosine (Tyr) at position 221, valine (Val) to Ile at position 272, alanine (Ala) to glutamic acid (Glu) at position 389, serine (Ser) to alanine (Ala) at position 395, valine (Val) to isoleucine (Ile) at position 410, and isoleucine (Ile) to valine (Val) at position 425. substitution of glycine (Gly) to alanine (Ala) at position 472; substitution of alanine (Ala) to serine (Ser) at position 607; substitution of isoleucine (Ile) to valine (Val) at position 623; substitution of arginine (Arg) to serine (Ser) at position 662; substitution of leucine (Leu) to glutamine (Gln) at position 670; substitution of threonine (Th) at position 699 substitution of asparagine (Asn) at position 765; substitution of aspartic acid (Asp) at position 707 with glycine (Gly); substitution of serine (Ser) at position 757 with alanine (Ala); substitution of glycine (Gly) at position 763 with serine (Ser); substitution of serine (Ser) at position 764 with asparagine (Asn); substitution of methionine (Met) at position 765 with valine (Val).

[0417] We investigated the relationship between the phages ΦXc10 and f20-Xaj, which had the highest similarity scores to the amino acid sequence of the tailtube protein of the phage isolated in Example 7. As a result, for tailtube protein A, SEQ ID NOs: 24 and 49 belong to the amino acid sequence shown in SEQ ID NO: 60, whereas no known phages, including ΦXc10 and f20-Xaj, belong to the amino acid sequence shown in SEQ ID NO: 60. Furthermore, for tailtube protein B, SEQ ID NOs: 58 and 50 belong to the amino acid sequence shown in SEQ ID NO: 61, whereas no known phages, including ΦXc10 and f20-Xaj, belong to the amino acid sequence shown in SEQ ID NO: 61.

[0418] As described above in Example 7, phages ΦXc10 and f20-Xaj did not exhibit lytic activity against Xanthomonas arboricola pv. pruni, suggesting that the lytic activity unique to phage No. 7 is due to differences in the amino acid sequence of the tailtube protein. Furthermore, even if there are slight differences in the amino acid sequence of the tailtube protein, as in the case of phage No. 7 obtained in this example, it was suggested that similar host ranges can be achieved depending on the position of the difference.

[0419] Example 13: Isolation of a novel ninth bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against pathogenic bacteria that cause plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0420] (Methods and Results) (1) Obtaining and Cultivating Plant Pathogenic Bacteria In this invention, the target plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the National Agriculture and Food Research Organization (NARO). The Xanthomonas bacteria listed in Table 13 were used in this example.

[0421] For control purposes, a strain of environmentally beneficial Pseudomonas fluorescens, which has been reported to have plant growth promoting effects, was obtained from NCIMB, a research institute within the UK National Culture Collection (UKNCC) (NCIMB-ID: 10460).

[0422] Various Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in Example 1, "(1) Obtaining and culturing plant pathogenic bacteria."

[0423] (2) Isolation and Purification of Phages The novel phage was isolated from natural wastewater, soil, or plant bodies (leaves) obtained in Japan. The isolation and purification methods were in accordance with the methods described in "(2) Isolation and Purification of First Phage" in Example 1. As a result, one new phage was isolated from natural wastewater, soil, or water in which plant bodies had been soaked.

[0424] The isolated phages were suspended in SM buffer and passed through a 0.2 μm filter to collect the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.

[0425] (3) Amplification and Purification of Phages The plate lysate (PL) method was carried out to amplify and purify the isolated and purified phages. The specific method was in accordance with the method described in "(3) Amplification and Purification of First Phage" in Example 1. The titer of the prepared purified phage solution was determined by plaque assay using an appropriately diluted solution. 8 It was confirmed that the PFU / mL or more.

[0426] (4) Evaluation of host range of phage The host range of the obtained phages was evaluated by a spot test method. The basic procedure was in accordance with the method described in "(4) Evaluation of host range of first phage" in Example 1.

[0427] An example of the results is shown in Figure 18. If a phage exhibits lytic activity, only the area of ​​the bacterial lawn formed on the plate where the phage purified solution was dropped will become transparent. The phage obtained in this example, like the phage obtained in Example 9, exhibited lytic activity against all strains belonging to three bacterial species: Xanthomonas arboricola, Xanthomonas citri, and Xanthomonas campestris (Figure 18). It was also confirmed that the phage exhibited lytic activity against strains of different pathogenic types. On the other hand, it was also confirmed that the phage did not exhibit lytic activity against Pseudomonas fluorescens. This suggests that the isolated phage may have a high degree of similarity to the ninth phage.

[0428] Furthermore, these results suggest that the phages obtained in this example are useful for controlling diseases caused by bacteria of the genus Xanthomonas, such as bacterial borer of peach, canker of citrus, and black rot of broccoli.

[0429] (5) Genome Analysis of Phages The genomic DNA sequences of the obtained phages were determined and analyzed.

[0430] (i) Preparation and sequencing of phage genomic DNA TURBO DNA-free TM The phage genome was extracted using a PCR amplification kit (Thermo Fisher Scientific) according to the basic procedure described in "(5) Genome analysis of first phage (i) Preparation and sequencing of genomic DNA of first phage" in Example 1.

[0431] (ii) Bioinformatics Analysis Based on Genome Sequence Information Based on the genome sequence (SEQ ID NO: 56) of the phage obtained in (i) above, the average nucleotide identity (ANI) to the genome sequence (SEQ ID NO: 41) of the ninth phage was analyzed using GENETYX-NGS. As a result, the sequence shown in SEQ ID NO: 56 had 92.61% sequence identity over a range of 75.06% to the sequence shown in SEQ ID NO: 41. It was unexpected that the identity of the genome sequence was not extremely high.

[0432] To clarify the cause of the identical host specificity between the phage isolated in this example and the phage isolated in Example 9, the sequences of the tailtube proteins of both phages were compared.

[0433] First, the gene cluster encoding the tailtube protein was identified from the genome sequence of the phage in this example. The BRAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ) were used to identify the genes.

[0434] The nucleotide sequence of the tailtube protein A gene contained in the genome nucleotide sequence of the phage discovered in this example (SEQ ID NO: 56) was completely identical to the nucleotide sequence of the tailtube protein A gene of the phage isolated in Example 9 (SEQ ID NO: 39). Therefore, the amino acid sequence of the translated tailtube protein A was identical to the amino acid sequence of the tailtube protein A of the phage isolated in Example 9 (SEQ ID NO: 37). For this reason, the phage obtained in this example and the phage obtained in Example 9 are referred to as the "ninth phage."

[0435] On the other hand, SEQ ID NO: 55 shows the nucleotide sequence of the tailtube protein B gene, and SEQ ID NO: 54 shows the amino acid sequence of tailtube protein B.

[0436] Furthermore, the amino acid sequence of tailtube protein B (SEQ ID NO:54) was compared with the amino acid sequence of tailtube protein B of the phage isolated in Example 9 (SEQ ID NO:38). The sequence identity of the amino acid sequence of tailtube protein B was 99.3%. Specifically, the amino acid sequence of SEQ ID NO:54 contained the following six amino acid substitutions compared to the amino acid sequence of SEQ ID NO:38: alanine (Ala) for threonine (Thr) at position 61, lysine (Lys) for glutamine (Gln) at position 108, glutamine (Gln) for proline (Pro) at position 404, serine (Ser) for proline (Pro) at position 679, alanine (Ala) for threonine (Thr) at position 682, and valine (Val) for isoleucine (Ile) at position 727.

[0437] As described above in Example 9, no known phages share 97% or more sequence identity with the amino acid sequences of tailtube proteins A and B of the ninth phage obtained in Example 9. Furthermore, phages such as Xanthomonas phage Xaa_vB_phi31, which had a high similarity score to the ninth phage, were not known to exhibit broad lytic activity against various species of Xanthomonas bacteria, as was the case with the ninth phage. This suggests that the broad lytic activity of the ninth phage is due to differences in the amino acid sequence of the tailtube protein. Furthermore, it suggests that phages with slight differences in the amino acid sequence of the tailtube protein, as in the case of the ninth phage obtained in this example, have a similar host range.

[0438] Example 14: Lytic activity of the obtained bacteriophage combinations (Objective) Novel bacteriophages that have been shown to have lytic activity when used alone against pathogenic bacteria that cause plant diseases were examined for their effectiveness against plant diseases when used in combination.

[0439] (Methods) (1) Spot test method A spot test was carried out according to the method described in "(4) Evaluation of host range of first phage" in Example 1. In this example, Xanthomonas arboricola pv. pruni (MAFF No. 311351), a bacterial strain for which the bacteriolytic activity of all tested phages has been confirmed, was used.

[0440] The phage purified solution to be dropped was a mixed solution prepared by mixing equal amounts of the phage purified solutions prepared in each Example. The mixed solution was prepared by diluting appropriately so that the total titer was equivalent to that when each phage was used alone. The phages used in this Example are as follows.

[0441] In this example, the following phage was used: a first phage having the genomic DNA sequence of SEQ ID NO: 8; a second phage having the genomic DNA sequence of SEQ ID NO: 13; a third phage having the genomic DNA sequence of SEQ ID NO: 14; a fourth phage having the genomic DNA sequence of SEQ ID NO: 17; a fifth phage having the genomic DNA sequence of SEQ ID NO: 18; a sixth phage having the genomic DNA sequence of SEQ ID NO: 23; a seventh phage having the genomic DNA sequence of SEQ ID NO: 28; an eighth phage having the genomic DNA sequence of SEQ ID NO: 32; a ninth phage having the genomic DNA sequence of SEQ ID NO: 41; and a tenth phage having the genomic DNA sequence of SEQ ID NO: 44.

[0442] (Results) Examples of results are shown in Figures 19 and 20. When a phage combination exhibits bacteriolytic activity, only the area of ​​the bacterial lawn formed on the plate where the purified phage solution was dropped becomes transparent. It was found that all of the tested combinations of two, five, and eight phages exhibited bacteriolytic activity at least as good as when each phage was used alone (Figures 19 and 20).

[0443] From the above results, it was confirmed that the phages of the present invention are useful whether used alone or in combination.

[0444] Example 15: Test of disease control effect against bacterial bore of peach (Objective) The effect of isolated novel bacteriophages having lytic activity against pathogenic bacteria of plant diseases on plant diseases when applied to plants was examined.

[0445] (Method) (1) Preparation of phage spray solution The phage spray solution used in this test was prepared as follows. First, a culture solution of the host bacteria for phage amplification was prepared as follows. The host used was Xanthomonas arboricola pv. pruni (MAFF No. 311351), a strain in which lytic activity has been confirmed for all tested phages. The bacterial cells were inoculated into YPG broth and incubated overnight in a shaker set at 25°C. After incubation, the OD 600 The turbidity (at a wavelength of 600 nm) was measured, and the one that reached about 1.0 was used as the bacterial culture solution below.

[0446] The prepared bacterial cell culture solution and the purified phage solution containing one type of phage prepared in Examples 1 to 13 (titer of 10 8 A mixture of equal amounts of phage (approximately PFU / mL) was inoculated into 100 times the volume of YPG culture medium and incubated for approximately 8-12 hours in a shaker set at 25°C. The resulting culture medium was recovered as a crude phage solution. One-tenth the volume of chloroform was added to the recovered crude solution, and after vigorously stirring, the solution was centrifuged at 8,000g / 20°C / 5 minutes to recover the supernatant. The recovered supernatant was passed through a 0.2 μm filter, and the filtrate was used as a purified phage solution.

[0447] The phage dispersion solution containing one type of phage was prepared by using a purified phage solution with a titer of approximately 10 9 The solution was diluted with sterile tap water to a concentration of approximately PFU / mL.

[0448] The phage dispersion solution containing multiple types of phages was prepared by mixing equal amounts of purified phage solutions adjusted to have the same titer. 9 The phage was diluted with sterile tap water to about PFU / mL and used. The first phage had the genomic DNA sequence of SEQ ID NO: 8, the second phage had the genomic DNA sequence of SEQ ID NO: 13, the third phage had the genomic DNA sequence of SEQ ID NO: 14, the fourth phage had the genomic DNA sequence of SEQ ID NO: 17, the fifth phage had the genomic DNA sequence of SEQ ID NO: 18, the sixth phage had the genomic DNA sequence of SEQ ID NO: 23, the seventh phage had the genomic DNA sequence of SEQ ID NO: 28, and the eighth phage had the genomic DNA sequence of SEQ ID NO: 32.

[0449] (2) Preparation of bacterial spray solution The bacterial spray solution used for infecting plants in this test was prepared using the bacterial culture solution prepared in (1). The bacterial culture solution was diluted approximately 10,000 times with sterile tap water and applied to a YPG agar plate, which was then incubated in an incubator set at 25°C for approximately 1 to 3 days. The bacterial suspension was collected by suspending colonies on the YPG agar plate in sterile tap water, and the final OD 600 The solution was diluted with sterilized tap water to a concentration of about 0.5 to prepare a bacterial spray solution.

[0450] (3) Plant Samples Commercially available peach seedlings (variety: Kawanakajima, 1 year old) were grown in a greenhouse, and seedlings grown to have about 100 leaves were used as samples for evaluation.

[0451] (4) Phage application and infection treatment. In the phage treatment group, each specimen was foliar-sprayed with the phage solution twice, twice before and twice after bacterial infection, with an interval of 2–3 days between each treatment. Two to three days later, the infected specimens were sprayed with the bacterial solution. The infected specimens were left in a greenhouse with high humidity for approximately two days after infection. Then, the phage solution was sprayed twice more on the leaves, with an interval of 2–3 days between each treatment. The untreated group was treated in the same manner as above, except that sterilized tap water was used instead of the purified phage solution.

[0452] (5) Measurement of Disease Incidence Rate One week or more after infection, when a certain level of disease incidence was confirmed in the untreated group, the disease incidence rates were investigated in the untreated group and each phage-sprayed group.

[0453] The leaves that showed brown spots characteristic of bacterial peach borer disease were judged to be diseased, and the ratio of diseased leaves to the total number of leaves was calculated as the disease incidence. The relative disease incidence in the phage-treated group was calculated as a relative value when the disease incidence in the untreated group was set at 100%.

[0454] (Results) The results are shown in Figures 21 and 22. The disease incidence in the untreated group was approximately 36%. On the other hand, the relative disease incidence, with the disease incidence in the untreated group taken as 100%, was approximately 50% on average when a phage spray containing one type of phage was applied. When phages were applied, the highest relative disease incidence was approximately 61% (the group applied with the sixth phage and the group applied with the eighth phage). On the other hand, the relative disease incidence was the lowest in the group applied with the seventh phage, at approximately 24%.

[0455] Furthermore, when a phage spray containing two or more phages was applied, the disease incidence was further reduced compared to when a phage spray containing a single phage was applied. The disease incidence in the untreated group was approximately 15%. When phages were applied in combination, the highest relative disease incidence was approximately 41% (combination of the second, fourth, sixth, and eighth phages). On the other hand, when the first and second phages were combined, the relative disease incidence was the lowest at approximately 34%.

[0456] From the above, it was found that the phages of the present invention can effectively control plants from diseases even when applied to actual plants. It was also found that even greater effects can be obtained by applying them in combination. Furthermore, it was found that the disease control effect of the phages of the present invention is effective even under conditions where the disease incidence rate is low in the untreated group.

[0457] Chemical pesticides containing common antibiotics can cause phytotoxicity to leaves, such as yellowing of the leaf tips. However, in the phage-treated group, no noticeable adverse effects on plants, such as phytotoxicity to leaves, which are thought to be caused by phages, were observed. This demonstrates that application of the phages of the present invention is an effective means of disease control with few side effects.

[0458] Example 16: Test of disease control effect against black rot of broccoli (Objective) The effect of isolated novel bacteriophages having lytic activity against pathogenic bacteria of plant diseases on plant disease when applied to plants was examined.

[0459] (Method) (1) Preparation of phage spray solution A phage spray solution was prepared in accordance with the description of Example 15, except that the bacterium used as the bacterial cell was Xanthomonas campestris pv. campestris (MAFF No. 106765) and the following phages were used:

[0460] In this example, a phage having the genomic DNA sequence of SEQ ID NO:7 (Φ1 in FIG. 23) was used as the first phage; a phage having the genomic DNA sequence of SEQ ID NO:13 (Φ2-1 in FIG. 23) and a phage having the genomic DNA sequence of SEQ ID NO:47 (Φ2-2 in FIG. 23) were used as the second phages; a phage having the genomic DNA sequence of SEQ ID NO:28 (Φ7-1 in FIG. 23) and a phage having the genomic DNA sequence of SEQ ID NO:53 (Φ7-2 in FIG. 23) were used as the seventh phages; a phage having the genomic DNA sequence of SEQ ID NO:41 (Φ9 in FIG. 23) was used as the ninth phage; and a phage having the genomic DNA sequence of SEQ ID NO:44 (Φ10 in FIG. 23) was used as the tenth phage.

[0461] (2) Preparation of bacterial spray solution A phage spray solution was prepared in accordance with the description of Example 15, except that the bacterial cells were the above-mentioned bacteria.

[0462] (3) Plant Samples Commercially available broccoli seeds were sown and grown in a greenhouse. Seedlings with five or more leaves were used as samples for evaluation.

[0463] (4) Phage application and infection treatment were carried out in accordance with the description in Example 15.

[0464] (5) Measurement of Disease Incidence The same procedure as in Example 15 was followed, except that the evaluation was carried out 16 days after infection.

[0465] (Results) The results are shown in Figure 23. The disease incidence in the untreated group was approximately 52%. On the other hand, the relative disease incidence, with the disease incidence in the untreated group taken as 100%, was approximately 62% on average when a phage spray containing one type of phage was applied. When phages were applied, the highest relative disease incidence was approximately 66% (the group in which the phage having the genomic DNA sequence of SEQ ID NO:28 (Φ7-1 in Figure 23) was applied as the seventh phage). On the other hand, the relative disease incidence was the lowest, at approximately 56%, in the group in which the phage having the genomic DNA sequence of SEQ ID NO:47 (Φ2-2 in Figure 23) was applied as the second phage.

[0466] These results confirmed that the ninth and tenth phages exhibit lytic activity even though the specific bacterial strains are different from the bacterial strains belonging to Xanthomonas campestris pv. campestris for which lytic activity was confirmed in Examples 9 and 10, respectively.

[0467] Furthermore, when a phage spray containing two or more phages was applied, the relative incidence rate further decreased to an average of approximately 53%. The highest relative incidence rate was approximately 58% when the ninth phage (Φ9 in Figure 23) and the tenth phage (Φ10 in Figure 23) were combined. On the other hand, the lowest relative incidence rate was approximately 47% when a total of four phages (Φ1 in Figure 23), two second phages (Φ2-1 and Φ2-2 in Figure 23), and the tenth phage (Φ10 in Figure 23) were combined.

[0468] From the above, it was found that the phages of the present invention can effectively control plants from diseases even when applied to actual plants, regardless of the type of plant itself. It was also found that a higher effect can be obtained by applying the phages of the present invention in combination.

[0469] In the phage-treated group, no noticeable adverse effects on the plants that are thought to be caused by the phages, such as phytotoxicity on the leaves, were observed. This demonstrates that application of the phages of the present invention is an effective means of disease control with few side effects.

[0470] Example 17: Test of disease control effect against bacterial spot of tomato (Objective) The effect of isolated novel bacteriophages having lytic activity against pathogenic bacteria of plant diseases on plant diseases when applied to plants was examined.

[0471] (Method) (1) Preparation of phage spray solution A phage spray solution was prepared in accordance with the description of Example 15, except that the bacterium used as the bacterial cell was Xanthomonas campestris pv. vesicatoria (MAFF No. 301256) and the following phages were used.

[0472] In this example, a phage having the genomic DNA sequence of SEQ ID NO: 7 (Φ1 in FIG. 24) was used as the first phage; a phage having the genomic DNA sequence of SEQ ID NO: 13 (Φ2-1 in FIG. 24), a phage having the genomic DNA sequence of SEQ ID NO: 47 (Φ2-2 in FIG. 24), and a phage having the genomic DNA sequence of SEQ ID NO: 48 (Φ2-3 in FIG. 24) were used as the second phages; a phage having the genomic DNA sequence of SEQ ID NO: 28 (Φ7 in FIG. 24) was used as the seventh phage; and a phage having the genomic DNA sequence of SEQ ID NO: 44 (Φ10 in FIG. 24) was used as the tenth phage.

[0473] (2) Preparation of bacterial spray solution A phage spray solution was prepared in accordance with the description of Example 15, except that the bacterial cells were the above-mentioned bacteria.

[0474] (3) Plant Samples Commercially available tomato seeds were sown and grown in a greenhouse. Seedlings with 50 or more leaves were used as samples for evaluation.

[0475] (4) Phage application and infection treatment were carried out in accordance with the description in Example 15.

[0476] (5) Measurement of Disease Incidence The same procedure as in Example 15 was followed, except that the evaluation was carried out 16 days after infection.

[0477] (Results) The results are shown in Figure 24. The disease incidence in the untreated group was approximately 25%. On the other hand, the relative disease incidence, with the disease incidence in the untreated group taken as 100%, was approximately 60% on average when a phage spray containing one type of phage was applied. When phages were applied, the highest relative disease incidence was approximately 66% (the group in which a phage having the genomic DNA sequence of SEQ ID NO: 47 (Φ2-2 in Figure 24) was applied as the second phage). On the other hand, the relative disease incidence was the lowest, at approximately 53%, in the group in which the seventh phage (Φ7 in Figure 24) was applied.

[0478] These results confirmed that the seventh and tenth phages exhibit lytic activity even though the specific bacterial strains are different from the bacterial strains belonging to Xanthomonas campestris pv. vesicatoria for which lytic activity was confirmed in Examples 12 and 10, respectively.

[0479] Furthermore, when a phage spray containing two or more phages was applied, the relative disease incidence rate further decreased to an average of approximately 49%. The highest relative disease incidence rate was approximately 55% when the second phage (Φ2-3 in Figure 24) having the genomic DNA sequence of SEQ ID NO:48 was combined with the seventh phage (Φ7 in Figure 24) as the second phage. On the other hand, the lowest relative disease incidence rate was approximately 43% when a total of four phages (Φ1 in Figure 24), consisting of the first phage (Φ1 in Figure 24), two second phages (Φ2-1 and Φ2-2 in Figure 24), and the tenth phage (Φ10 in Figure 24) were combined.

[0480] From the above, it was found that the phages of the present invention can effectively control plants from diseases even when applied to actual plants, regardless of the type of plant itself. It was also found that a higher effect can be obtained by applying the phages of the present invention in combination.

[0481] In the phage-treated group, no noticeable adverse effects on the plants that could be attributed to the phages, such as phytotoxicity to the leaves, were observed. This demonstrates that application of the phages of the present invention is an effective means of disease control with few side effects. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A bacteriophage comprising a bacteriophage whose genomic DNA contains a gene encoding a tail fiber protein having recognition activity for target bacteria, and whose amino acid sequence is as shown in any one of (a) to (d) below: (a) an amino acid sequence represented by any one selected from the group consisting of SEQ ID NOs: 11, 45, 42, and 1, (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: 42 or 1, (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 42 or 1, or (d) An amino acid sequence in which one amino acid other than those at positions 278 and 350 is substituted in the amino acid sequence shown in Sequence ID No.

11.

2. A bacteriophage comprising a bacteriophage containing in its genomic DNA a gene comprising the amino acid sequence shown in any one of (e) to (g) below, and a gene comprising the amino acid sequence shown in any one of (h) to (j) below, wherein the genes are a gene encoding tail tube protein A and a gene encoding tail tube protein B, respectively, which have recognition activity for target bacteria: (e) The amino acid sequence shown in SEQ ID NOs: 24, 49, or 60, (f) Amino acid sequences in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 24 or 49, (g) an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in Sequence ID No. 24 or 49, (h) Amino acid sequence shown in SEQ ID NO: 57, 50, or 61, (i) 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: 57 or 50, (j) An amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in Sequence ID No. 57 or 50.

3. The genomic DNA contains a gene consisting of the amino acid sequence shown in any one of (k) to (m) below, and a gene consisting of the amino acid sequence shown in any one of (n) to (p) below. A lytic agent comprising a bacteriophage, wherein the genes are a gene encoding tail tube protein A and a gene encoding tail tube protein B, respectively, which have recognition activity for target bacteria: (k) The amino acid sequence shown in Sequence ID No. 37, (l) 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. 37, (m) An amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in Sequence ID No. 37, (n) Amino acid sequence shown in Sequence ID No. 38, 54, or 59, (o) 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: 38 or 54, or (p) An amino acid sequence having 92% or more sequence identity with the amino acid sequence shown in Sequence ID No. 38 or 54.

4. The lytic agent according to claim 1, wherein the gene consists of a base sequence shown in any one of the following (1) to (3): (1) A base sequence shown by any one selected from the group consisting of SEQ ID NOs: 12, 46, 43 and 5-7, (2) A nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted in any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs. 43 and 5-7, or (3) A nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by any one selected from the group consisting of Sequence ID No. 43 and 5-7.

5. The lytic agent according to claim 4, wherein the base sequence of the genomic DNA consists of any one of the following base sequences (1') to (7'): (1') A base sequence shown by any one selected from the group consisting of SEQ ID NOs: 13, 47, 48, 44 and 8-10, (2') A nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted to a nucleotide sequence other than the gene described in claim 4, in which a nucleotide sequence represented by any one selected from the group consisting of sequence numbers 13, 47, 48, 44 and 8 to 10, (3') A base sequence in which any one selected from the group consisting of Sequence ID No. 44 and 8 to 10 has 80% or more sequence identity with the base sequences other than the gene described in claim 4, (4') A nucleotide sequence in which the nucleotide sequence other than the gene described in claim 4 has 98.5% or more sequence identity, (5') A nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted in any one selected from the group consisting of SEQ ID NOs: 13, 47, 48, 44 and 8-10, or (6') A nucleotide sequence having 90% or more sequence identity in any one selected from the group consisting of Sequence ID No. 44 and 8-10, (7') A nucleotide sequence having 98.5% or more sequence identity with the nucleotide sequence shown in SEQ ID NOs: 13, 47, or 48.

6. The lytic agent according to claim 2, wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (8') to (12') below: (8') The base sequence shown in Sequence ID No. 28, 29 or 53, (9') A nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted to a nucleotide sequence other than the gene described in claim 2, in the nucleotide sequence shown in sequence number 28, 29, or 53, (10') A base sequence in which the base sequence other than the gene described in claim 2 has 95% or more sequence identity, (11') A nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 28, 29, or 53, or (12') A nucleotide sequence having 95% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 28, 29, or 53.

7. The lytic agent according to claim 3, wherein the base sequence of the genomic DNA consists of any one of the base sequences shown in (13') to (17') below: (13') The base sequence shown in SEQ ID NO: 41 or 56, (14') A nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted to a nucleotide sequence other than the gene described in claim 3, in the nucleotide sequence shown in sequence number 41 or 56, (15') A base sequence in which a base sequence other than the gene described in claim 3 has 80% or more sequence identity, (16') A nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 41 or 56, or (17') A nucleotide sequence having 90% or more sequence identity in the nucleotide sequence shown in Sequence ID No. 41 or 56.

8. A lytic agent according to claim 1, which exhibits lytic activity against bacteria of the genus Xanthomonas.

9. A composition comprising one or more lysing agents as described in any one of claims 1 to 8 as active ingredients.

10. A plant disease control composition comprising the composition described in claim 9.

11. The plant disease control composition according to claim 10, which is a plant disease caused by bacteria of the genus Xanthomonas.

12. 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 10.

13. A method for identifying bacteria of the genus Xanthomonas, A culture process to obtain a culture by culturing test bacteria isolated from plant tissue affected by plant diseases. A mixing step of mixing the culture with the lysant according to any one of claims 1 to 8 to obtain a mixture, A mixture culture step in which the mixture is cultured under predetermined conditions, and A determination step in which, if the test bacteria are lysed after the mixture culture step, the test bacteria are determined to be of the genus Xanthomonas. The method comprising the above.

14. The method according to claim 13, 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.

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