Xanthomonas bacteria lysogenic bacteriophage

Abstract

In order to control plant diseases caused by Xanthomonas bacteria, the present invention develops and provides a plant disease control composition in which a novel bacteriophage that specifically exhibits lysolytic activity with respect to the bacteria of the genus is isolated and contains the same as an active ingredient. Provided are: a bacteriolytic agent which has a novel genomic DNA sequence and contains a bacteriophage that specifically exhibits bacteriolytic activity against Xanthomonas bacteria; and a plant disease control composition which contains the bacteriolytic agent as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a bacteriolytic agent containing bacteriophage, a plant disease control composition containing the bacteriolytic agent, and a plant disease control method. Background Art

[0002] Bacteriophage (sometimes simply referred to as "phage" in this specification) is a general term for viruses that infect only bacteria. Most phages, called lytic phages, specifically adsorb to target bacteria as hosts, inject their own DNA, and self-amplify using the bacterial translation mechanism. Furthermore, the amplified phages are spread by lysing the bacteria, repeatedly infecting new target bacteria (Non-Patent Document 1).

[0003] It is known that most of the bacteria of the genus Xanthomonas are pathogens of plant diseases represented by various crops, and copper agents and antibiotics are used as general routine control. However, there are many problems such as drug efficacy, phytotoxicity, and even becoming the main cause of imbalance of bacterial flora. Therefore, in recent years, the method of applying bacteriophage has attracted much attention as a new control method (Non-Patent Document 2).

[0004] Phages showing lytic activity against Xanthomonas bacteria are reported in Patent Documents 1 to 3 and Non-Patent Document 2. However, since the host range of phages is extremely narrow, the search for new phages and the development of phages with higher lytic activity are still important.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2016-32435

[0008] Patent Document 2: Japanese Patent Application Publication No. 2021-102635

[0009] Patent Document 3: International Publication No. 2017 / 113029

[0010] Non-patent literature

[0011] Non-patent literature 1: Sharma S. et al., Folia Microbiol., 2017, 62: 17-55

[0012] Non-patent literature 2: Nakayinga R. et al., BMC Microbiology, 2021, 21: 291 Summary of the invention

[0013] Problems to be solved by the invention

[0014] In order to solve the above problems, the present inventors have focused on bacteriophages. Unlike existing copper agents and antibiotics, bacteriophages, which are viruses, are natural products, and there has been no report on obvious drug damage so far. In addition, since the specificity for the host is very high, only bacteria of a specific genus or species can become targets, and the impact on the balance of the flora is extremely limited. In addition, it is harmless not only to animals represented by humans, but also to plants, and has high safety.

[0015] Therefore, in order to suppress the damage to crops caused by plant diseases caused by Xanthomonas bacteria, a new bacteriophage showing lytic activity against Xanthomonas bacteria was isolated. In addition, the present invention aims to provide a composition containing the bacteriophage as an active ingredient, and to use the composition for disease control, pathogenic bacteria detection, etc.

[0016] Solution to the problem

[0017] The present inventors isolated a new phage from natural sewage / soil using a method for detecting lytic plaques formed on a soft agar medium cultured with Xanthomonas bacteria, evaluated the lytic activity of the phage against various Xanthomonas bacteria, and analyzed its genome sequence. The results showed that it was a phage having a new genome DNA sequence. The present invention was completed based on the above research and development results, and specifically provides the following aspects.

[0018] (1-1) A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a tail fiber protein having recognition activity for a target bacterium and having an amino acid sequence as shown in any one of the following (a) to (c): (a) the amino acid sequence shown in sequence number 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 sequence number 1; (c) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in sequence number 1.

[0019] (1-2) The bacteriolytic agent according to (1-1), wherein the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence represented by any one of SEQ ID NOs: 2 to 4.

[0020] (1-3) A bacteriolytic agent according to (1-1) or (1-2), wherein the gene comprises a base sequence as shown in any one of the following (d) to (f): (d) a base sequence as shown in any one of sequence numbers 5 to 7; (e) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in any one of sequence numbers 5 to 7; (f) a base sequence having a sequence identity of 90% or more with the base sequence as shown in any one of sequence numbers 5 to 7.

[0021] (1-4) The bacteriolytic agent according to any one of (1-1) to (1-3), wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (g) to (k): (g) a base sequence represented by any one of SEQ ID NOs: 8 to 10; (h) a base sequence represented by any one of SEQ ID NOs: 8 to 10, in which one or more bases are added, deleted and / or substituted to a base sequence other than the base sequence of the gene described in any one of (1-1) to (1-3); (i) a base sequence represented by any one of SEQ ID NOs: 8 to 10, in which one or more bases are added, deleted and / or substituted to a base sequence other than the base sequence of the gene described in any one of (1-1) to (1-3); (i) a base sequence represented by any one of SEQ ID NOs: 8 to 10, in which one or more bases are added, deleted and / or substituted to a base sequence described in any one of SEQ ID NOs: 8 to 10; (k) a base sequence represented by any one of SEQ ID NOs: 8 to 10, in which one or more bases are added, deleted and / or substituted to a base sequence described in any one of SEQ ID NOs: 8 to 10.

[0022] (1-5) The bacteriolytic agent according to any one of (1-1) to (1-4), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0023] (1-6) A lytic 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.

[0024] (2-1) A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a tail fiber protein having recognition activity for a target bacterium and an amino acid sequence represented by any one of the following (a) or (b): (a) an amino acid sequence represented by sequence number 11 or 45, or (b) an amino acid sequence in which one amino acid other than positions 278 and 350 is substituted in the amino acid sequence represented by sequence number 11.

[0025] (2-2) The bacteriolytic agent according to (2-1), wherein the gene comprises the base sequence represented by SEQ ID NO: 12 or 46.

[0026] (2-3) The bacteriolytic agent according to (2-1) or (2-2), wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (a) to (e): (a) a base sequence represented by sequence number 13, 47 or 48; (b) a base sequence represented by sequence number 13, 47 or 48 in which one or more bases are added, deleted and / or substituted in a base sequence other than the gene base sequence described in (2-1) or (2-2). ; (c) a base sequence having a sequence identity of 98.5% or more with a base sequence other than the gene base sequence described in (2-1) or (2-2) above in the base sequence shown in sequence numbers 13, 47 or 48; (d) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence numbers 13, 47 or 48; (e) a base sequence having a sequence identity of 98.5% or more with the base sequence shown in sequence numbers 13, 47 or 48.

[0027] (2-4) The bacteriolytic agent according to any one of (2-1) to (2-3), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0028] (2-5) A lytic agent according to (2-4), 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.

[0029] (3-1) A lytic agent comprising a bacteriophage having a genomic DNA sequence comprising a base sequence as shown in any one of the following (a) to (c): (a) a base sequence as shown in SEQ ID NO: 14; (b) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in SEQ ID NO: 14; (c) a base sequence having a sequence identity of 90% or more with the base sequence as shown in SEQ ID NO: 14.

[0030] (3-2) The bacteriolytic agent according to (3-1), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0031] (3-3) The bacteriolytic agent according to (3-2), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0032] (4-1) A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a tail fiber protein having recognition activity for a target bacterium and having an amino acid sequence as shown in any one of the following (a) to (c): (a) an amino acid sequence as shown in sequence number 15; (b) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in sequence number 15; (c) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence as shown in sequence number 15.

[0033] (4-2) A lytic agent according to (4-1), wherein the gene comprises a base sequence as shown in any one of the following (d) to (f): (d) a base sequence shown in sequence number 16; (e) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 16; (f) a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence number 16.

[0034] (4-3) The bacteriolytic agent according to (4-1) or (4-2), wherein the base sequence of the genomic DNA comprises a base sequence as shown in any one of the following (g) to (k): (g) a base sequence as shown in SEQ ID NO: 17; (h) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence other than the base sequence of the gene as shown in (4-1) or (4-2); (i) a base sequence having a sequence identity of 80% or more with a base sequence in the base sequence as shown in SEQ ID NO: 17 other than the base sequence of the gene as shown in (4-1) or (4-2); (i) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in SEQ ID NO: 17; (k) a base sequence having a sequence identity of 90% or more with the base sequence as shown in SEQ ID NO: 17.

[0035] (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.

[0036] (4-5) The bacteriolytic agent according to (4-4), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0037] (5-1) A lytic agent comprising a bacteriophage having a genomic DNA sequence comprising a base sequence as shown in any one of the following (a) to (c): (a) a base sequence as shown in sequence number 18 or 19; (b) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in sequence number 18 or 19; (c) a base sequence having a sequence identity of 90% or more with the base sequence as shown in sequence number 18 or 19.

[0038] (5-2) The bacteriolytic agent according to (5-1), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0039] (5-3) The bacteriolytic agent according to (5-2), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0040] (6-1) A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a protein having an amino acid sequence as shown in any one of the following (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 21; (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: 21; (c) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 21.

[0041] (6-2) A lytic agent according to (6-1), wherein the gene comprises a base sequence as shown in any one of the following (d) to (f): (d) a base sequence shown in sequence number 22; (e) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 22; (f) a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence number 22.

[0042] (6-3) A lytic agent according to (6-1) or (6-2), wherein the base sequence of the genomic DNA comprises a base sequence as shown in any one of the following (g) to (k): (g) a base sequence as shown in SEQ ID NO: 23; (h) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence other than the gene as shown in (6-1) or (6-2) in the base sequence as shown in SEQ ID NO: 23; (i) a base sequence having a sequence identity of 95% or more with a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence other than the gene as shown in (6-1) or (6-2) in the base sequence as shown in SEQ ID NO: 23; (k) a base sequence having a sequence identity of 95% or more with the base sequence as shown in SEQ ID NO: 23.

[0043] (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.

[0044] (6-5) The bacteriolytic agent according to (6-4), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0045] (7-1) A lytic agent comprising a bacteriophage, wherein the phage comprises in its genomic DNA: a gene encoding a tail tube protein A having a recognition activity for a target bacterium and comprising an amino acid sequence as shown in any one of the following (a) to (c), and a gene encoding a tail tube protein B having a recognition activity for a target bacterium and comprising an amino acid sequence as shown in any one of the following (d) to (f): (a) an amino acid sequence as shown in SEQ ID NO: 24, 49 or 60; (b) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in SEQ ID NO: 24 or 49; (c) an amino acid sequence having a sequence identity of 97% or more with the amino acid sequence as shown in SEQ ID NO: 24 or 49; (d) an amino acid sequence as shown in SEQ ID NO: 57, 50 or 61; (e) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in SEQ ID NO: 57 or 50; (f) an amino acid sequence having a sequence identity of 97% or more with the amino acid sequence as shown in SEQ ID NO: 57 or 50.

[0046] (7-2) A lytic agent according to (7-1), wherein the gene encoding the above-mentioned tail tube protein A contains a base sequence shown in any one of the following (g) to (i): (g) a base sequence shown in sequence number 26 or 51; (h) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 26 or 51; (i) a base sequence having a sequence identity of more than 97% with the base sequence shown in sequence number 26 or 51.

[0047] (7-3) A lytic agent according to (7-1) or (7-2), wherein the gene encoding the above-mentioned tail tube protein B contains a base sequence shown in any one of the following (j) to (1): (j) a base sequence shown in sequence number 27 or 52; (k) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 27 or 52; (l) a base sequence having a sequence identity of more than 97% with the base sequence shown in sequence number 27 or 52.

[0048] (7-4) A bacteriolytic agent according to any one of (7-1) to (7-3), wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (m) to (q): (m) a base sequence represented by sequence number 28, 29 or 53; (n) a base sequence represented by sequence number 28, 29 or 53 in which one or more bases are added, deleted and / or substituted in a base sequence other than the gene base sequence described in any one of (7-1) to (7-3). (o) a base sequence having a sequence identity of 95% or more to a base sequence other than the gene described in any one of (7-1) to (7-3) in the base sequence shown in sequence numbers 28, 29 or 53; (p) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence numbers 28, 29 or 53; (q) a base sequence having a sequence identity of 95% or more to the base sequence shown in sequence numbers 28, 29 or 53.

[0049] (7-5) The bacteriolytic agent according to any one of (7-1) to (7-4), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0050] (7-6) The bacteriolytic agent according to (7-5), wherein the Xanthomonas bacterium is Xanthomonas arboricola or Xanthomonas campestris.

[0051] (8-1) A bacteriolytic agent comprising a bacteriophage,

[0052] The bacteriophage has a genomic DNA sequence comprising a base sequence shown in any one of the following (a) to (c): (a) a base sequence shown in any one of sequence numbers 32 to 36; (b) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 32 to 36; (c) a base sequence having a sequence identity of more than 90% with the base sequence shown in any one of sequence numbers 32 to 36.

[0053] (8-2) The bacteriolytic agent according to (8-1), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0054] (8-3) The bacteriolytic agent according to (8-2), wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0055] (9-1) A lytic agent comprising a bacteriophage, wherein the phage comprises in its genomic DNA: a gene comprising an amino acid sequence as shown in any one of the following (a) to (c) and encoding a tail tube protein A having recognition activity for a target bacterium, and a gene comprising an amino acid sequence as shown in any one of the following (d) to (f) and encoding a tail tube protein B having recognition activity for a target bacterium: (a) an amino acid sequence as shown in SEQ ID NO: 37; (b) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in SEQ ID NO: 37; (c) an amino acid sequence having a sequence identity of 92% or more with the amino acid sequence as shown in SEQ ID NO: 37; (d) an amino acid sequence as shown in SEQ ID NO: 38, 54 or 59; (e) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in SEQ ID NO: 38 or 54; (f) an amino acid sequence having a sequence identity of 92% or more with the amino acid sequence as shown in SEQ ID NO: 38 or 54.

[0056] (9-2) A lytic agent according to (9-1), wherein the gene encoding the above-mentioned tail tube protein A contains a base sequence shown in any one of the following (g) to (i): (g) a base sequence shown in sequence number 39; (h) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 39; (i) a base sequence having a sequence identity of more than 90% with the base sequence shown in sequence number 39.

[0057] (9-3) A lytic agent according to (9-1) or (9-2), wherein the gene encoding the above-mentioned tail tube protein B contains a base sequence shown in any one of the following (j) to (l): (j) a base sequence shown in sequence number 40 or 55; (k) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 40 or 55; (l) a base sequence having a sequence identity of more than 90% with the base sequence shown in sequence number 40 or 55.

[0058] (9-4) A lytic agent according to any one of (9-1) to (9-3), wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (m) to (q): (m) a base sequence represented by SEQ ID NO: 41 or 56; (n) a base sequence represented by SEQ ID NO: 41 or 56 in which one or more bases are added, deleted and / or substituted to a base sequence other than the base sequence of the gene described in any one of (9-1) to (9-3); (o) a base sequence having a sequence identity of 80% or more with a base sequence represented by SEQ ID NO: 41 or 56 other than the gene described in any one of (9-1) to (9-3); (p) a base sequence represented by one or more bases are added, deleted and / or substituted to a base sequence represented by SEQ ID NO: 41 or 56; (q) a base sequence having a sequence identity of 90% or more with the base sequence represented by SEQ ID NO: 41 or 56.

[0059] (9-5) The bacteriolytic agent according to any one of (9-1) to (9-4), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0060] (9-6) The bacteriolytic agent according to (9-5), wherein the Xanthomonas bacterium is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, and Xanthomonas citri.

[0061] (10-1) A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a tail fiber protein having recognition activity for a target bacterium and having an amino acid sequence as shown in any one of the following (a) to (c): (a) an amino acid sequence as shown in sequence number 42; (b) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in sequence number 42; (c) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence as shown in sequence number 42.

[0062] (10-2) A lytic agent according to (10-1), wherein the gene comprises a base sequence as shown in any one of the following (d) to (f): (d) a base sequence shown in sequence number 43; (e) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 43; (f) a base sequence having a sequence identity of more than 90% with the base sequence shown in sequence number 43.

[0063] (10-3) A bacteriolytic agent according to (10-1) or (10-2), wherein the base sequence of the genomic DNA comprises a base sequence as shown in any one of the following (g) to (k): (g) a base sequence as shown in sequence number 44; (h) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence other than the base sequence of the gene as shown in (10-1) or (10-2); (i) a base sequence having a sequence identity of 80% or more with a base sequence in the base sequence as shown in sequence number 44 other than the base sequence of the gene as shown in (10-1) or (10-2); (i) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in sequence number 44; (k) a base sequence having a sequence identity of 90% or more with the base sequence as shown in sequence number 44.

[0064] (10-4) The bacteriolytic agent according to any one of (10-1) to (10-3), which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0065] (10-5) The bacteriolytic agent according to (10-4), wherein the Xanthomonas bacterium is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campe stris, and Xanthomonas citri.

[0066] [1-1] A bacteriolytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a tail fiber protein having recognition activity for a target bacterium and containing an amino acid sequence as shown in any one of the following (a) to (d): (a) an amino acid sequence selected from any one of sequence numbers 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 sequence number 42 or 1, (c) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in sequence number 42 or 1, or (d) an amino acid sequence in which one amino acid other than positions 278 and 350 is substituted in the amino acid sequence shown in sequence number 11.

[0067] [1-2] A bacteriolytic agent comprising a bacteriophage, wherein the phage comprises in its genomic DNA a gene containing an amino acid sequence as shown in any one of the following (e) to (g), and a gene containing an amino acid sequence as shown in any one of the following (h) to (j), wherein the above genes are respectively a gene encoding a tail tube protein A having recognition activity for a target bacterium and a gene encoding a tail tube protein B: (e) an amino acid sequence as shown in SEQ ID NO: 24, 49 or 60, (f) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in SEQ ID NO: 24 or 49, (g) an amino acid sequence having a sequence identity of 97% or more with the amino acid sequence as shown in SEQ ID NO: 24 or 49, (h) an amino acid sequence as 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 as shown in SEQ ID NO: 57 or 50, or (j) an amino acid sequence having a sequence identity of 97% or more with the amino acid sequence as shown in SEQ ID NO: 57 or 50.

[0068] [1-3] A lytic agent comprising a bacteriophage, wherein the phage comprises in its genomic DNA a gene containing an amino acid sequence as shown in any one of the following (k) to (m), and a gene containing an amino acid sequence as shown in any one of the following (n) to (p), wherein the above genes are respectively a gene encoding a tail tube protein A having recognition activity for a target bacterium and a gene encoding a tail tube protein B: (k) the amino acid sequence shown in SEQ ID NO: 37, (1) 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: 37, (m) an amino acid sequence having a sequence identity of 92% or more with the amino acid sequence shown in SEQ ID NO: 37, (n) an amino acid sequence shown in SEQ 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 a sequence identity of 92% or more with the amino acid sequence shown in SEQ ID NO: 38 or 54.

[0069] [1-4] The bacteriolytic agent according to [1-1], wherein the amino acid sequence represented by the above-mentioned sequence number 1 is an amino acid sequence represented by any one of sequence numbers 2 to 4.

[0070] [1-5] The bacteriolytic agent according to [1-1], wherein the gene comprises a base sequence represented by any one of the following (1) to (3): (1) a base sequence selected from the group consisting of SEQ ID NOs: 12, 46, 43 and 5 to 7, (2) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence selected from the group consisting of SEQ ID NOs: 43 and 5 to 7, or (3) a base sequence having a sequence identity of 90% or more with the base sequence selected from the group consisting of SEQ ID NOs: 43 and 5 to 7.

[0071] [1-6] The bacteriolytic agent according to [1-5], wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (1') to (7'): (1') a base sequence selected from any one of SEQ ID NOs: 13, 47, 48, 44 and 8 to 10, (2') a base sequence selected from any one of SEQ ID NOs: 13, 47, 48, 44 and 8 to 10 in which one or more bases are added, deleted and / or substituted in a base sequence other than the gene described in [1-5], (3') a base sequence selected from any one of SEQ ID NOs: 44 and 8 to 10 in which the base sequence other than the gene described in [1-5] has 80 % or more, (4') a base sequence having a sequence identity of 98.5% or more with the base sequence of the base sequence shown in SEQ ID NOs: 13, 47 or 48 except the above-mentioned gene described in [1-5], (5') a base sequence in which one or more bases are 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 a sequence identity of 90% or more with the base sequence shown in any one of SEQ ID NOs: 44 and 8 to 10, (7') a base sequence having a sequence identity of 98.5% or more with the base sequence shown in SEQ ID NOs: 13, 47 or 48.

[0072] [1-7] A lytic agent according to [1-2], wherein the gene encoding the tail tube protein A comprises a base sequence shown in any one of the following (4) to (6): (4) a base sequence shown in sequence number 26 or 51, (5) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 26 or 51, or (6) a base sequence having a sequence identity of 97% or more with the base sequence shown in sequence number 26 or 51.

[0073] [1-8] A lytic agent according to [1-2] or [1-7], wherein the gene encoding the above-mentioned tail tube protein B contains a base sequence shown in any one of the following (7) to (9): (7) a base sequence shown in sequence number 27 or 52, (8) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 27 or 52, or (9) a base sequence having a sequence identity of 97% or more with the base sequence shown in sequence number 27 or 52.

[0074] [1-9] The bacteriolytic agent according to [1-2], wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (8') to (12'): (8') a base sequence represented by SEQ ID NO: 28, 29 or 53, (9') a base sequence represented by SEQ ID NO: 28, 29 or 53 in which one or more bases are added, deleted and / or substituted for a base sequence other than the gene described in [1-2], (10') a base sequence having a sequence identity of 95% or more with a base sequence represented by SEQ ID NO: 28, 29 or 53 in which one or more bases are added, deleted and / or substituted for a base sequence other than the gene described in [1-2], (11') a base sequence represented by SEQ ID NO: 28, 29 or 53 in which one or more bases are added, deleted and / or substituted for a base sequence, or (12') a base sequence having a sequence identity of 95% or more with a base sequence represented by SEQ ID NO: 28, 29 or 53.

[0075] [1-10] A lytic agent according to [1-3], wherein the gene encoding the tail tube protein A comprises a base sequence as shown in any one of the following (10) to (12): (10) a base sequence as shown in sequence number 39, (11) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in sequence number 39, or (12) a base sequence having a sequence identity of 90% or more with the base sequence as shown in sequence number 39.

[0076] [1-11] A lytic agent according to [1-3] or [1-10], wherein the gene encoding the above-mentioned tail tube protein B contains a base sequence shown in any one of the following (13) to (15): (13) a base sequence shown in sequence number 40 or 55, (14) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 40 or 55, or (15) a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence number 40 or 55.

[0077] [1-12] The bacteriolytic agent according to [1-3], wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (13') to (17'): (13') a base sequence represented by SEQ ID NO: 41 or 56, (14') a base sequence represented by SEQ ID NO: 41 or 56 in which one or more bases are added, deleted and / or substituted for a base sequence other than the gene described in [1-3], (15') a base sequence having a sequence identity of 80% or more with a base sequence represented by SEQ ID NO: 41 or 56 in which one or more bases are added, deleted and / or substituted for a base sequence other than the gene described in [1-3], (16') a base sequence represented by SEQ ID NO: 41 or 56 in which one or more bases are added, deleted and / or substituted for a base sequence, or (17') a base sequence having a sequence identity of 90% or more with a base sequence represented by SEQ ID NO: 41 or 56.

[0078] [1-13] The bacteriolytic agent according to any one of [1-1] to [1-12], which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0079] [2-1] A bacteriolytic agent comprising a bacteriophage, wherein the phage comprises in its genomic DNA a gene containing an amino acid sequence as shown in any one of the following (a) to (c), and a gene containing an amino acid sequence as shown in any one of the following (d) to (f), wherein the above genes are respectively a gene encoding a tail tube protein A and a gene encoding a tail tube protein B having recognition activity for a target bacterium: (a) an amino acid sequence as shown in SEQ ID NO: 24, 49 or 60, (b) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in SEQ ID NO: 24 or 49, (c) an amino acid sequence having a sequence identity of 97% or more with the amino acid sequence as shown in SEQ ID NO: 24 or 49, (d) an amino acid sequence as shown in SEQ ID NO: 57, 50 or 61, (e) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence as shown in SEQ ID NO: 57 or 50, or (f) an amino acid sequence having a sequence identity of 97% or more with the amino acid sequence as shown in SEQ ID NO: 57 or 50.

[0080] [2-2] A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a tail fiber protein having recognition activity for a target bacterium and containing an amino acid sequence as shown in any one of the following (g) to (j): (g) an amino acid sequence selected from any one of SEQ ID NOs: 1, 11, 45, 15 and 42, (h) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence selected from any one of SEQ ID NOs: 1, 11, 15 and 42, (i) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence selected from any one of SEQ ID NOs: 1, 11, 15 and 42, or (j) an amino acid sequence in which one amino acid other than positions 278 and 350 is substituted in the amino acid sequence selected from SEQ ID NO: 11.

[0081] [2-3] A lytic agent comprising a bacteriophage, wherein the phage comprises in its genomic DNA a gene containing an amino acid sequence as shown in any one of the following (k) to (m), and a gene containing an amino acid sequence as shown in any one of the following (n) to (p), wherein the above genes are respectively a gene encoding a tail tube protein A having recognition activity for a target bacterium and a gene encoding a tail tube protein B: (k) the amino acid sequence shown in SEQ 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 SEQ ID NO: 37, (m) an amino acid sequence having a sequence identity of 92% or more with the amino acid sequence shown in SEQ ID NO: 37, (n) an amino acid sequence shown in SEQ 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 a sequence identity of 92% or more with the amino acid sequence shown in SEQ ID NO: 38 or 54.

[0082] [2-4] A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a protein having an amino acid sequence represented by any one of the following (q) to (s): (q) the amino acid sequence represented by SEQ ID NO: 21, (r) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence represented by SEQ ID NO: 21, or (s) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 21.

[0083] [2-5] A lytic agent comprising a bacteriophage, wherein the bacteriophage has a genomic DNA sequence comprising a base sequence represented by any one of the following (1') to (3'): (1') a base sequence selected from any one of SEQ ID NOs: 14, 18, 19 and 32 to 36, (2') a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence selected from any one of SEQ ID NOs: 14, 18, 19 and 32 to 36, or (3') a base sequence having a sequence identity of 90% or more with the base sequence selected from any one of SEQ ID NOs: 14, 18, 19 and 32 to 36.

[0084] [2-6] The bacteriolytic agent according to [2-1], wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (4') to (8'): (4') a base sequence represented by SEQ ID NO: 28, 29 or 53, (5') a base sequence represented by SEQ ID NO: 28, 29 or 53 in which one or more bases are added, deleted and / or substituted for a base sequence other than the gene described in [2-2], (6') a base sequence having a sequence identity of 95% or more with a base sequence represented by SEQ ID NO: 28, 29 or 53 in which one or more bases are added, deleted and / or substituted for a base sequence other than the gene described in [2-2], (7') a base sequence represented by SEQ ID NO: 28, 29 or 53 in which one or more bases are added, deleted and / or substituted for a base sequence, or (8') a base sequence having a sequence identity of 95% or more with a base sequence represented by SEQ ID NO: 28, 29 or 53.

[0085] [2-7] The bacteriolytic agent according to any one of [2-1] to [2-6], which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0086] [3-1] A lytic agent comprising a bacteriophage having a genomic DNA sequence comprising a base sequence represented by any one of the following (1') to (3'): (1') a base sequence represented by any one of sequence numbers 32 to 36, 14, 18 and 19, (2') a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence represented by any one of sequence numbers 32 to 36, 14, 18 and 19, or (3') a base sequence having a sequence identity of 90% or more with the base sequence represented by any one of sequence numbers 32 to 36, 14, 18 and 19.

[0087] [3-2] A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a tail fiber protein having recognition activity for a target bacterium and having an amino acid sequence as shown in any one of the following (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 15, (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: 15, or (c) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 15.

[0088] [3-3] A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene encoding a protein having an amino acid sequence as shown in any one of the following (d) to (f): (d) the amino acid sequence shown in SEQ ID NO: 21, (e) 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: 21, or (f) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 21.

[0089] [3-4] A bacteriolytic agent according to [3-2], wherein the gene comprises a base sequence represented by any one of the following (1) to (3): (1) a base sequence represented by sequence number 16, (2) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence represented by sequence number 16, or (3) a base sequence having a sequence identity of 90% or more with the base sequence represented by sequence number 16.

[0090] [3-5] The bacteriolytic agent according to [3-2], wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (4') to (8'): (4') the base sequence represented by SEQ ID NO: 17, (5') a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence represented by SEQ ID NO: 17 other than the gene represented by SEQ ID NO: 3-2, (6') a base sequence having a sequence identity of 80% or more with the base sequence in the base sequence represented by SEQ ID NO: 17 other than the gene represented by SEQ ID NO: 3-2, (7') a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence represented by SEQ ID NO: 17, or (8') a base sequence having a sequence identity of 90% or more with the base sequence represented by SEQ ID NO: 17.

[0091] [3-6] A bacteriolytic agent according to [3-3], wherein the gene comprises a base sequence as shown in any one of the following (4) to (6): (4) a base sequence as shown in sequence number 22, (5) a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in sequence number 22, or (6) a base sequence having a sequence identity of 90% or more with the base sequence as shown in sequence number 22.

[0092] [3-7] The bacteriolytic agent according to [3-3], wherein the base sequence of the genomic DNA comprises a base sequence represented by any one of the following (9') to (13'): (9') the base sequence represented by SEQ ID NO: 23, (10') a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence represented by SEQ ID NO: 23, (11') a base sequence having a sequence identity of 95% or more with the base sequence in the base sequence represented by SEQ ID NO: 23 except the base sequence represented by SEQ ID NO: 3-3, (12') a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence represented by SEQ ID NO: 23, or (13') a base sequence having a sequence identity of 95% or more with the base sequence represented by SEQ ID NO: 23.

[0093] [3-8] The bacteriolytic agent according to any one of [3-1] to [3-7], which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0094] [3-9] The bacteriolytic agent according to [3-8], wherein the Xanthomonas bacterium is Xanthomonas arboricola.

[0095] [3-10] The bacteriolytic agent according to [3-9], wherein the pathotype of the Xanthomonas bacteria is pruni.

[0096] [4-1] A bacteriolytic agent comprising a bacteriophage, wherein the bacteriophage contains in its genomic DNA a gene that contains an amino acid sequence selected from any one of SEQ ID NOs: 42, 11, 45, and 15 and encodes a tail fiber protein having a recognition activity for a target bacterium.

[0097] [4-2] The bacteriolytic agent according to [4-1], wherein the gene comprises a base sequence selected from any one of SEQ ID NOs: 43, 12, 46 and 16.

[0098] [4-3] The bacteriolytic agent according to [4-1], wherein the base sequence of the genomic DNA comprises a base sequence selected from any one of SEQ ID NOs: 44, 13, 47, 48 and 17.

[0099] [4-4] A bacteriolytic agent comprising a bacteriophage, wherein the phage contains a gene in its genomic DNA that contains the amino acid sequence shown in SEQ ID NO: 1 and encodes a tail fiber protein having a recognition activity for a target bacterium.

[0100] [4-5] The bacteriolytic agent according to [4-4], wherein the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence represented by any one of SEQ ID NOs: 2 to 4.

[0101] [4-6] The bacteriolytic agent according to [4-4], wherein the gene comprises the base sequence represented by any one of SEQ ID NOs: 5 to 7.

[0102] [4-7] The bacteriolytic agent according to [4-4], wherein the base sequence of the genomic DNA comprises the base sequence represented by any one of SEQ ID NOs: 8 to 10.

[0103] [4-8] A bacteriolytic agent comprising a bacteriophage having a genomic DNA sequence comprising a base sequence selected from the group consisting of SEQ ID NOs: 14, 18, 19, and 32 to 36.

[0104] [4-9] A bacteriolytic agent comprising a bacteriophage containing a gene encoding a protein containing the amino acid sequence shown in SEQ ID NO: 21 in its genomic DNA.

[0105] [4-10] The bacteriolytic agent according to [4-9], wherein the gene comprises the base sequence shown in SEQ ID NO: 22.

[0106] [4-11] The bacteriolytic agent according to [4-9], wherein the base sequence of the genomic DNA includes the base sequence shown in SEQ ID NO: 23.

[0107] [4-12] A lytic agent comprising a bacteriophage, wherein the phage contains in its genomic DNA a gene comprising an amino acid sequence represented by sequence numbers 24, 49 or 37 and encoding a tail tube protein A having recognition activity for a target bacterium, and a gene comprising an amino acid sequence represented by any one of sequence numbers 57, 50, 38, 54 and 59 and encoding a tail tube protein B having recognition activity for a target bacterium.

[0108] [4-13] The bacteriolytic agent according to [4-12], wherein the gene encoding the tail tube protein A comprises the base sequence shown in SEQ ID NO: 26, 51 or 39.

[0109] [4-14] The bacteriolytic agent according to [4-12], wherein the gene encoding the tail tube protein B comprises a base sequence selected from any one of SEQ ID NOs: 27, 52, 40 and 55.

[0110] [4-15] The bacteriolytic agent according to [4-12], wherein the base sequence of the genomic DNA comprises a base sequence selected from any one of SEQ ID NOs: 28, 29, 53, or 41.

[0111] [4-16] The bacteriolytic agent according to any one of [4-1] to [4-15], which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas.

[0112] <1> A composition comprising one or more bacteriolytic agents according to any one of (1-1) to (10-5) and [1-1] to [4-16] as an active ingredient.

[0113] <2> A composition comprising two or more bacteriolytic agents according to any one of (1-1) to (10-5) and [1-1] to [4-16] as active ingredients.

[0114] <3> A plant disease control composition comprising <1> or <2> The composition.

[0115] <4> according to <3> The plant disease control composition is a plant disease control composition for plant diseases caused by bacteria of the genus Xanthomonas.

[0116] <5> according to <3> or <4> The plant disease control composition comprises other bacteriophages showing bacteriolytic activity against Xanthomonas bacteria.

[0117] <6> A method for preventing and controlling plant diseases, comprising:

[0118] Contact process, <3> ~ <5> The plant disease control composition described in any one of the above is contacted with a target plant.

[0119] <7> A method for identifying Xanthomonas bacteria, the method comprising:

[0120] a culturing step of culturing the test bacteria separated from the plant tissue infected by the plant disease to obtain a culture;

[0121] a mixing step of mixing the culture with the bacteriolytic agent described in any one of (1-1) to (10-5) and [1-1] to [4-16] to obtain a mixture;

[0122] a mixture culturing step, culturing the mixture under given conditions; and

[0123] A determination step of determining that the test bacteria are Xanthomonas bacteria when the test bacteria undergo lysis after the mixture culturing step.

[0124] <8> according to <7> The method, wherein in the mixture culturing step, the mixture further comprises a liquid culture medium containing soft agar, and the mixture is cultured on a solid culture medium.

[0125] <9> according to <7> The method, wherein in the culturing step, the culture comprises a liquid culture medium containing soft agar, and the culture is cultured on a solid culture medium.

[0126] <10> according to <7> ~ <9> The method described in any one of the above, further comprising before the above culturing step:

[0127] The isolation step is to isolate the test bacteria from the plant tissues infected by the plant disease.

[0128] This specification includes the disclosure 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 as the basis of the priority of the present application.

[0129] Effects of the Invention

[0130] The bacteriolytic agent of the present invention and the composition containing the same as an active ingredient can lyse specific target bacteria.

[0131] According to the plant disease control composition of the present invention, diseases caused by specific target bacteria can be prevented and suppressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 : are graphs showing the bacteriolytic activity of the first phage obtained in Example 1. A is a graph showing that the Xanthomonas bacteria shown in Table 2 and the control Pseudomonas fluore scens were cultured on an agar plate, and then the first phage purified solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the phage purified solution dropped onto each plate. Figure 1In A and B, 1 represents a plate on which bacteria with ID MAFF No.211191 were developed, 2 represents a plate on which bacteria with ID MAFF No.311351 were developed, 3 represents a plate on which bacteria with ID MAFF No.301256 were developed, 4 represents a plate on which bacteria with ID MAFF No.106765 were developed, 5 represents a plate on which bacteria with ID MAFF No.301078 were developed, and 6 represents a plate on which Pseudomonas fluorescens for control with ID NCIMB-ID 10460 was developed. Figure 1 In B, a represents the position of the purified solution of the phage having the genomic DNA sequence of sequence number 8, b represents the position of the purified solution of the phage having the genomic DNA sequence of sequence number 9, and c represents the position of the purified solution of the phage having the genomic DNA sequence of sequence number 10.

[0133] Figure 2 : are graphs showing the bacteriolytic activity of the second phage obtained in Example 2. A is a graph showing that after Xanthomonas bacteria and Pseudomonas fiuorescens for control were cultured on an agar plate, the purified second phage solution was dropped onto the center of the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the bacteria shown in Table 4 developed on each plate.

[0134] Figure 3 : are graphs showing the bacteriolytic activity of the third phage obtained in Example 3. A is a graph showing that the Xanthomonas bacteria shown in Table 5 and the control Pseudomonas fluore scens were cultured on an agar plate, and then the third phage purified solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the phage purified solution dropped onto each plate. Figure 3 In A and B, 1 represents a plate on which bacteria with ID MAFF No.311282 were developed, 2 represents a plate on which bacteria with ID MAFF No.301426 were developed, 3 represents a plate on which bacteria with ID MAFF No.311351 were developed, 4 represents a plate on which bacteria with ID MAFF No.311586 were developed, 5 represents a plate on which bacteria with ID MAFF No.311618 were developed, and 6 represents a plate on which Pseudomonas fluorescens for control with ID NCIMB-ID 10460 was developed.

[0135] Figure 4Graphs showing the bacteriolytic activity of the fourth phage obtained in Example 4. A is a graph showing that after Xanthomonas bacteria and Pseudomonas fluorescens for control were cultured on an agar plate, the purified solution of the fourth phage was dropped onto the center of the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the bacteria shown in Table 6 developed on each plate.

[0136] Figure 5 This is a graph showing the bacteriolytic activity of the fifth phage obtained in Example 5. Figure 5 The upper part (1 to 3) of the graph shows the lytic activity of the phage of sequence number 18, and the lower part (4 to 6) shows the lytic activity of the phage of sequence number 19. A is a graph showing that the bacteria of the genus Xanthomonas shown in Table 7 and the control Pseudomonas fluorescens were cultured on an agar plate, and then the purified solution of the fifth phage was dripped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the purified solution of phage dripped onto each plate. Figure 5 In A and B, 1 and 4 indicate plates on which bacteria with ID MAFF No. 211971 were developed, 2 and 5 indicate plates on which bacteria with ID MAFF No. 311351 were developed, and 3 and 6 indicate plates on which Pseudomonas fluorescens for control with ID NCIMB-ID 10460 was developed.

[0137] Figure 6 Graphs showing the bacteriolytic activity of the sixth phage obtained in Example 6. A is a graph showing that after Xanthomonas bacteria and Pseudomonas fluorescens for control were cultured on an agar plate, the purified solution of the sixth phage was dropped onto the center of the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the bacteria shown in Table 9 developed on each plate.

[0138] Figure 7 Graphs showing the bacteriolytic activity of the seventh phage obtained in Example 7. A is a graph showing that the bacteria of the genus Xanthomonas shown in Table 10 and the control Pseudomonas fluorescens were cultured on an agar plate, and then the purified solution of the seventh phage was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the purified phage solution dropped onto each plate. Figure 7In A and B, 1 indicates a plate on which bacteria with ID MAFF No. 311351 were developed, 2 indicates a plate on which bacteria with ID MAFF No. 311622 were developed, 3 indicates a plate on which bacteria with ID MAFF No. 106642 were developed, and 4 indicates a plate on which Pseudomo nas fluorescens for control with ID NCIMB-ID 10460 was developed. In Figure B, a indicates the position of a purified solution of a phage having a genomic DNA sequence of SEQ ID No. 28, and b indicates the position of a purified solution of a phage having a genomic DNA sequence of SEQ ID No. 29.

[0139] Figure 8 : are graphs showing the bacteriolytic activity of the 8th phage obtained in Example 8. A is a graph showing that the Xanthomonas bacteria shown in Table 11 and Pseudomonas fluorescens for control were cultured on an agar plate, and then the purified 8th phage solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the purified phage solution dropped onto each plate. Figure 8 In A and B, 1 indicates a plate on which bacteria with ID MAFF No.211971 were developed, 2 indicates a plate on which bacteria with ID MAFF No.311282 were developed, 3 indicates a plate on which bacteria with ID MAFF No.301420 were developed, 4 indicates a plate on which bacteria with ID MAFF No.301426 were developed, 5 indicates a plate on which bacteria with ID MAFF No.311351 were developed, 6 indicates a plate on which bacteria with ID MAFF No.311414 were developed, 7 indicates a plate on which bacteria with ID MAFF No.311417 were developed, 8 indicates a plate on which bacteria with ID MAFF No.311562 were developed, 9 indicates a plate on which bacteria with ID MAFF No.311571 were developed, and 10 indicates a plate on which bacteria with ID MAFF No.311572 were developed. 11 is a plate on which bacteria of ID MAFF No. 311618 were developed, 12 is a plate on which Pseudomonas fluorescens for control with ID NCIMB-ID 10460 was developed. Figure 8 In B, a indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO:32.

[0140] Fig. 9: are graphs showing the bacteriolytic activity of the 8th phage obtained in Example 8. A is a graph showing that the Xanthomonas bacteria shown in Table 11 and Pseudomonas fluorescens for control were cultured on an agar plate, and then the purified 8th phage solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the purified phage solution dropped onto each plate. Fig. 9 In A and B, 1 represents a plate on which bacteria with ID MAFF No.211971 were developed, 2 represents a plate on which bacteria with ID MAFF No.311282 were developed, 3 represents a plate on which bacteria with ID MAFF No.301420 were developed, 4 represents a plate on which bacteria with ID MAFF No.301426 were developed, 5 represents a plate on which bacteria with ID MAFF No.311351 were developed, and 6 represents a plate on which Pseudomonas fluorescens for control with ID NCIMB-ID 10460 was developed. Fig. 9 In B, b represents the position of the purified solution of the phage having the genomic DNA sequence of sequence number 33, c represents the position of the purified solution of the phage having the genomic DNA sequence of sequence number 34, d represents the position of the purified solution of the phage having the genomic DNA sequence of sequence number 35, and e represents the position of the purified solution of the phage having the genomic DNA sequence of sequence number 36.

[0141] Fig.10 Graphs showing the bacteriophage lytic activity obtained in Example 9. A is a graph showing that the Xanthomonas bacteria shown in Table 13 and Pseudomonas fluorescens for control were cultured on an agar plate, and then the purified phage solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the purified phage solution dropped onto each plate.

[0142] Fig.11 Graphs showing the bacteriophage lytic activity obtained in Example 10. A is a graph showing that the Xanthomonas bacteria shown in Table 13 and Pseudomonas fluorescens for control were cultured on an agar plate, and then the purified phage solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the purified phage solution dropped onto each plate.

[0143] Fig.12 This is a diagram showing an example of a plate for plaque assay of the first phage obtained in Example 1.

[0144] Fig.13 This is a diagram showing an example of a plate for plaque assay of the second phage obtained in Example 2.

[0145] Fig.14 : are graphs showing the bacteriolytic activity of the second phage obtained in Example 11. A is a graph showing that the Xanthomonas bacteria shown in Table 4 and the control Pseudomonas fluorescens were cultured on an agar plate, and then the second phage purified solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the position of the phage purified solution dropped onto each plate. Fig.14 In A and B, 1 indicates a plate on which bacteria with ID MAFF No. 673005 were developed, 2 indicates a plate on which bacteria with ID MAFF No. 301256 were developed, 3 indicates a plate on which bacteria with ID MAFF No. 301352 were developed, 4 indicates a plate on which bacteria with ID MAFF No. 212146 were developed, 5 indicates a plate on which bacteria with ID MAFF No. 311351 were developed, and 6 indicates a plate on which Pseudomonas fluorescens for control with ID NCIMB-ID 10460 was developed. In Figure B, a indicates the position of a purified solution of a phage having the genomic DNA sequence of SEQ ID No. 47, and b indicates the position of a purified solution of a phage having the genomic DNA sequence of SEQ ID No. 48.

[0146] Fig.15 Graphs showing the lytic activity of the seventh phage obtained in Example 2 and the two second phages obtained in Example 11. A is a graph showing that after the Xanthomonas bacteria shown in Table 14 were developed on an agar plate and cultured, the second phage purified solution was dripped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the positions of the phage purified solutions dripped onto each plate. In graph B, a indicates the position of the purified solution of the phage having the genomic DNA sequence of sequence number 12, b indicates the position of the purified solution of the phage having the genomic DNA sequence of sequence number 47, and c indicates the position of the purified solution of the phage having the genomic DNA sequence of sequence number 48.

[0147] Fig.16 : is a graph showing the bacteriolytic activity of the seventh phage obtained in Example 12. A is a graph showing that the Xanthomonas bacteria shown in Table 10 and the control Pseudomonas fluorescens were cultured on an agar plate, and then the phage purification solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the ID of the bacteria cultured on each plate.

[0148] Fig.17Graphs showing the bacteriolytic activity of the two seventh phages obtained in Example 7 and the phage obtained in Example 12. A is a graph showing that after the Xanthomonas bacteria shown by ID MAFF No. 301260 developed on an agar plate were cultured, the phage purified solution was dripped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the positions of the phage purified solution dripped onto each plate. In Figure B, a indicates the position of the purified solution of the phage having the genomic DNA sequence of sequence number 28, b indicates the position of the purified solution of the phage having the genomic DNA sequence of sequence number 29, and c indicates the position of the purified solution of the phage having the genomic DNA sequence of sequence number 53 obtained in Example 12.

[0149] Fig.18 : is a graph showing the bacteriolytic activity of the 9th phage obtained in Example 13. A is a graph showing the Xanthomonas bacteria shown in Table 13 developed on an agar plate and the phage purification solution was dropped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the ID of the bacteria cultured on each plate.

[0150] Fig.19 Graphs showing the bacteriolytic activity of the phage combinations tested in Example 14. A is a graph showing that after the Xanthomonas bacteria shown by ID MAFF No. 311351 were developed on an agar plate and cultured, a phage purification solution was dripped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the types of phages contained in the phage purification solution dripped onto each plate. In Figure B, a represents the 1st phage having a genomic DNA sequence of sequence number 8, b represents the 2nd phage having a genomic DNA sequence of sequence number 13, c represents the 7th phage having a genomic DNA sequence of sequence number 28, d represents the 9th phage having a genomic DNA sequence of sequence number 41, e represents the 10th phage having a genomic DNA sequence of sequence number 44, f represents the 3rd phage having a genomic DNA sequence of sequence number 14, g represents the 4th phage having a genomic DNA sequence of sequence number 17, h represents the 5th phage having a genomic DNA sequence of sequence number 18, i represents the 6th phage having a genomic DNA sequence of sequence number 23, and i represents the 8th phage having a genomic DNA sequence of sequence number 32. In the figure, " / " indicates that the phages before and after it are used in combination. For example, "a / b" means that the 1st phage is used in combination with the 2nd phage.

[0151] Fig. 20Graphs showing the bacteriolytic activity of the phage combinations tested in Example 14. A is a graph showing that after the Xanthomonas bacteria shown by ID MAFF No. 311351 were developed on an agar plate and cultured, a phage purification solution was dripped onto the plate and static culture was performed. B is a graph of the plate corresponding to A, showing the types of phages contained in the phage purification solution dripped onto each plate. In Figure B, a represents the 1st phage having a genomic DNA sequence of sequence number 8, b represents the 2nd phage having a genomic DNA sequence of sequence number 13, c represents the 7th phage having a genomic DNA sequence of sequence number 28, d represents the 9th phage having a genomic DNA sequence of sequence number 41, e represents the 10th phage having a genomic DNA sequence of sequence number 44, f represents the 3rd phage having a genomic DNA sequence of sequence number 14, g represents the 4th phage having a genomic DNA sequence of sequence number 17, h represents the 5th phage having a genomic DNA sequence of sequence number 18, i represents the 6th phage having a genomic DNA sequence of sequence number 23, and i represents the 8th phage having a genomic DNA sequence of sequence number 32. In the figure, " / " indicates that the phages before and after it are used in combination. For example, "f / g" means that the 3rd phage is used in combination with the 4th phage.

[0152] Fig.21 This is a graph showing the results of the disease control effect test of peach bacterial puncture in Example 15. The average incidence rate is expressed as a relative value relative to the untreated group. The numerical value shown below each bar graph represents the type of bacteriophage used. In each experimental group, the first to eighth phages were used alone.

[0153] Fig. 22 1. It is a graph showing the results of the disease control effect test of peach bacterial puncture disease tested in Example 15. The average incidence rate is expressed as a relative value relative to the untreated group. The numerical values ​​shown below each bar graph indicate the types of bacteriophages used in combination. In each experimental group, a combination of four bacteriophages consisting of the first phage, the third phage, the fifth phage, and the seventh phage; a combination of four bacteriophages consisting of the second phage, the fourth phage, the sixth phage, and the eighth phage; and a combination of eight phages consisting of the first phage to the eighth phage were used.

[0154] Fig.23It is a graph showing the results of the disease control effect test of cauliflower black rot tested in Example 16. The average incidence rate is expressed as a relative value relative to the untreated group. The numerical values ​​shown in each bar graph represent the type of bacteriophage used. In the figure, a circle indicates that the bacteriophage is used, and "-" indicates that the bacteriophage is not used. Respectively, Φ1 represents the 1st bacteriophage having a genomic DNA sequence of sequence number 10, Φ2-1 represents the 2nd bacteriophage having a genomic DNA sequence of sequence number 13, ΦΦ2-2 represents the 2nd bacteriophage having a genomic DNA sequence of sequence number 47, Φ7-1 represents the 7th bacteriophage having a genomic DNA sequence of sequence number 28, Φ7-2 represents the 7th bacteriophage having a genomic DNA sequence of sequence number 53, Φ9 represents the 9th bacteriophage having a genomic DNA sequence of sequence number 41, and Φ10 represents the 10th bacteriophage having a genomic DNA sequence of sequence number 44.

[0155] Fig.24 : This is a graph showing the results of the disease control effect test of bacterial spot disease of tomatoes tested in Example 17. The average incidence rate is expressed as a relative value relative to the untreated group. The numerical values ​​shown in each bar graph represent the type of bacteriophage used. In the figure, a circle indicates that the bacteriophage is used, and "-" indicates that the bacteriophage is not used. Respectively, Φ1 represents the 1st bacteriophage having a genomic DNA sequence of sequence number 10, Φ2-1 represents the 2nd bacteriophage having a genomic DNA sequence of sequence number 13, Φ2-2 represents the 2nd bacteriophage having a genomic DNA sequence of sequence number 47, Φ2-3 represents the 2nd phage having a genomic DNA sequence of sequence number 48, Φ7 represents the 7th bacteriophage having a genomic DNA sequence of sequence number 28, and Φ10 represents the 10th phage having a genomic DNA sequence of sequence number 44. DETAILED DESCRIPTION

[0156] 1. Lytic agent

[0157] 1-1. Overview

[0158] A first aspect of the present invention is a bacteriolytic agent. The bacteriolytic agent of the present invention contains a bacteriophage having a genome sequence containing a specific base sequence.

[0159] The bacteriolytic agent according to the present invention exhibits bacteriolytic activity against target bacteria that may become pathogenic bacteria causing plant diseases.

[0160] 1-2. Definition

[0161] The terms used in this specification are defined as follows.

[0162] In the present specification, the "bacteriolytic agent" refers to an agent containing a bacteriophage having bacteriolytic activity against a target bacterium.

[0163] "Bacteria" is one of the main systems of organisms that divide the entire biological kingdom into three parts, along with archaea and eukaryotes. Bacteria are composed of cells without a nucleus and can replicate themselves as long as there is a nutrient source. The names of bacteria are based on the International Code of Bacterial Nomenclature, and are expressed as genus and species under family.

[0164] In this specification, "target bacteria" refers to the phage constituting the bacteriolytic agent of the present invention, or the host bacteria that can become the target of the phage contained in the composition and plant disease control composition of the present invention. Specifically, for example, it is a bacterium having a membrane surface receptor on the outer membrane of the cell that is recognized by the above-mentioned phage. Or, for example, it is a bacterium having a membrane surface receptor on the outer membrane of the cell that is recognized by a tail fiber protein containing a specific amino acid sequence. In addition, it is a bacterium having a membrane surface receptor on the outer membrane of the cell that is recognized by a tail tube protein containing a specific amino acid sequence. "Membrane surface receptors" are binding sites of bacteriophages, such as the tail and tail fibers, and are composed of proteins, lipopolysaccharides or cilia, etc., present in the outer layer of the bacterial outer membrane. As specific examples of target bacteria in this specification, Xanthomonas bacteria can be cited.

[0165] "Xanthomonas bacteria" refers to bacteria belonging to the genus Xanthomonas. Xanthomonas bacteria generally produce yellow pigments such as xanthomonadin, and most of them are known as plant pathogenic bacteria. It should be noted that as a classification lower than species, there are subspecies or pathovar, which are indicated by adding subsp. or pv. after the bacterial name. In addition, the smallest unit of classification is strain, which refers to a group of cells that are considered to be genetically uniform. The following Table 1 shows representative Xanthomonas bacteria, their host plants, and plant diseases caused by them.

[0166] [Table 1]

[0167]

[0168]

[0169] Although not limited, among the bacteria of the genus Xanthomonas, Xanthomonas arboricola, Xanthomonas campestris, Xanthomonas citri, Xanthomonas oryzae, and Xanthomonas cucurbitae are preferably used as the target bacteria of the present invention. Specific examples of Xanthomonas arboricola include Xanthomonas arboricola pv. pruni, whose pathotype is pruni, and Xanthomonas arboricola pv. juglandis, whose pathotype is jugla ndis. Specific examples of Xanthomonas campestris include Xanthomonas campestris pv. vesicatoria, Xanthomonas campestris pv. campestris, Xanthomonas campestris pv. vitians, and Xanthomonas campestris pv. raphani. Specific examples of Xanthomonas citri include Xanthomonas citri subsp. citri. Specific examples of Xanthomonas oryzae include Xanthomonas oryzae pv. oryzae.

[0170] "Bacteriophage" (as mentioned above, sometimes simply referred to as "bacteriophage" in this specification) refers to the general term for viruses that infect bacteria. A general bacteriophage is composed of three parts: a head, a tail, and a tail fiber. The head is composed of capsomeres, which are capsid proteins, and contains a capsid (virus shell) with an icosahedral structure, in whose internal space the genomic DNA of the bacteriophage is built. The tail has a tubular structure formed by a tail tube protein and a sheath protein that coats the tail tube protein. One end of the tail is connected to the head, and the other end is connected to the tail fiber. The tail functions as an introduction tube for injecting the genomic DNA of the head into the cell of the host bacterium. The tail fiber is composed of several fiber structures containing tail fiber proteins. The tail and tail fibers recognize receptors present on the outer membrane surface of the host bacteria, and play a host recognition function and an adsorption function of adsorbing on the cell surface. The host specificity of the bacteriophage is extremely high, and its characteristics are based on the functions of the tail and tail fibers.

[0171] Bacteriophages do not infect eukaryotic organisms, and therefore, drugs using bacteriophages are harmless to humans, animals, and plants. It should be noted that bacteriophages can be roughly divided into "lytic cycle", "lysogenic cycle" and "lytic / lysogenic cycle" based on the mode of infection. In the lysogenic cycle, the target bacteria are not lysed, but their own DNA is introduced into the chromosome of the bacteria, and proliferates together with the proliferation of the bacteria. On the other hand, in the lytic cycle, after self-proliferation in the cells of the host bacteria, the host bacteria are lysed, releasing a large number of daughter phages. The object of the bacteriophage of the present invention is a virulent phage that has undergone a lytic cycle.

[0172] As mentioned above, "tail fiber protein" refers to the protein that constitutes the tail fiber of the bacteriophage. Tail fiber protein is known to play an important role in the specificity of host recognition and adsorption ability of the tail and tail fiber (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16: 760-773). Therefore, for new bacteriophages with characteristics on tail fiber protein, even if the host bacteria are the same as the known bacteriophages, due to the different host recognition sites, even bacteria with infection tolerance to known bacteriophages can show lytic activity, etc., and its utilization value is very high.

[0173] The "fiber gene" refers to a gene encoding the above-mentioned fiber protein contained in the genomic DNA of a bacteriophage.

[0174] As mentioned above, "tail tube protein" is a protein that constitutes the tubular structure of the tail of the bacteriophage. It is known that the tail tube protein interacts with the tail filament, and together with the tail filament, it plays an important role in the specificity of host recognition and adsorption ability (Maozhi Hu, et ai., 2020, 9: 1, 855-867). As tail tube proteins, tail tube fiber protein A and tail tube protein B are known. "Tail tube protein A" refers to a protein that forms a ring at the lower part of the tubular structure of the tail and interacts with the tail filament. "Tail tube protein B" refers to 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 bacteria.

[0175] The "tail tube gene" refers to a gene encoding the above-mentioned tail tube protein contained in the genomic DNA of the bacteriophage. The "tail tube protein A gene" refers to a gene encoding the tail tube protein A, and the "tail tube protein B gene" refers to a gene encoding the tail tube protein B.

[0176] "Bacteriolysis" refers to the phenomenon of destroying the cell membrane of bacteria. As mentioned above, it is mainly observed in the infection mode of virulent bacteriophages. Bacteria die due to bacteriolysis. Bacteriolysis starts with the phage specifically adsorbing to the target bacteria and injecting its own DNA into the target bacterial cells through the tail. Then, it replicates itself using the bacterial translation mechanism, produces a large number of daughter phages, and then lyses the bacteria to release the daughter phages to the outside world.

[0177] In this specification, "plant disease" refers to a general term for diseases that occur in plants. Among known plant diseases, in addition to diseases caused by infectious pathogens such as viruses, bacteria, filamentous fungi, actinomycetes, viroids, phytoplasmas, nematodes, mites or insects, there are also diseases caused by non-infectious pathogens such as deficiency or excess of nutrients or water, and pesticide damage. Unless otherwise specified, the plant diseases in this specification refer to diseases caused by bacteria, that is, plant pathogenic bacteria. In this specification, unless otherwise specified, plant pathogenic bacteria are equivalent to the above-mentioned target bacteria, such as Xanthomonas bacteria.

[0178] In this specification, "control" means prevention or treatment (expel) (according to the homepage of the Japan Agricultural Chemicals Industry Association). Therefore, in this specification, "plant disease control" means prevention of plant diseases, especially target bacteria, or treatment of plant diseases caused by target bacteria.

[0179] In this specification, "target plants" refer to plants to which the plant disease control composition of the present invention described below is applied. Such plants correspond to plants that have developed specific plant diseases due to infection with the target bacteria described above, or plants that are at risk of infection by the target bacteria.

[0180] 1-3. Composition

[0181] The bacteriolytic agent of the present invention comprises a bacteriophage.

[0182] <1st phage>

[0183] The first bacteriophage is characterized in that it contains a gene encoding a tail fiber protein containing a specific amino acid sequence in its genomic DNA and exhibits specific bacteriolytic activity against target bacteria.

[0184] (1-1) Tail fiber protein

[0185] The tail fiber protein comprises: an amino acid sequence shown in sequence number 1 consisting of 1371 amino acid residues, an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in sequence number 1, or an amino acid sequence having an amino acid sequence identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more with the amino acid sequence shown in sequence number 1. It is characterized in that any tail fiber protein has target bacteria-specific recognition activity.

[0186] It should be noted that, in the present specification, "plurality" refers to 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, 2 to 3. In addition, "(amino acid) substitution" refers to substitution within a conservative amino acid group with similar properties such as charge, branching, polarity, and aromaticity among the 20 amino acids constituting natural proteins. Examples include substitutions within the uncharged polar amino acid group with low polarity branches (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), the branched amino acid group (Leu, Val, Ile), the neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro), the neutral amino acid group with hydrophilic branches (Asn, Gln, Thr, Ser, Tyr, Cys), the acidic amino acid group (Asp, Glu), the basic amino acid group (Arg, Lys, His), and the aromatic amino acid group (Phe, Tyr, Trp). It is known that substitution of amino acids within these groups is unlikely to change the properties of the polypeptide and is therefore preferred.

[0187] In addition, in this specification, "amino acid (sequence identity)" refers to the numerical value of the ratio of the same type of amino acid residues within the comparison range of two amino acid sequences. When the lengths of the two amino acid sequences are different, the amino acid sequence identity can be calculated by matching (arranging) in a manner that the amino acid consistency within the comparison range is the highest. A representative algorithm for performing such an analysis is BLAST, but there is no limitation. BLAST can be used through various software and Web services. For example, the amino acid sequence identity can be easily calculated using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ), NCBI provides a BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), etc. In addition, in addition to BLAST, there is also an algorithm called FASTA, which can be used as long as the appropriate identity can be calculated.

[0188] The present inventors have discovered three phages that have lytic activity against Xanthomonas bacteria, and have identified the tail fiber genes from the genomic DNA sequences of these phages (sequence numbers 8 to 10, respectively). The amino acid sequences encoded by these tail fiber genes (sequence numbers 2 to 4) were analyzed using the genetic information processing software GEN ETYX, and the results showed that they had high sequence identity of more than 98% with each other. Sequence number 1 is an amino acid sequence common to the amino acid sequences of sequence numbers 2 to 4.

[0189] Therefore, the amino acid sequence represented by SEQ ID NO: 1 may be the amino acid sequence represented by any one of SEQ ID NOs: 2 to 4.

[0190] (1-2) Tail fiber gene

[0191] The first bacteriophage contains a fiber gene having a base sequence encoding the fiber protein in the genomic DNA of the bacteriophage.

[0192] As specific base sequences of the tail fiber gene, for example, there can be listed: the base sequence shown in any one of sequence numbers 5 to 7, or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 5 to 7, and a base sequence having a sequence identity of more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the base sequence shown in any one of sequence numbers 5 to 7, or a base sequence that hybridizes with a base sequence complementary to the base sequence shown in any one of sequence numbers 5 to 7 under highly stringent conditions.

[0193] In this specification, "base (sequence identity)" refers to the numerical value of the ratio of the same position of the type of base within the comparison range of two base sequences as the above-mentioned amino acid sequence identity. When the lengths of the two base sequences are different, the base sequence identity can be calculated by matching (arranging) in a manner that the base consistency within the comparison range reaches the highest. In addition to the above-mentioned BLAST, FASTA, the analysis of base sequence identity can also be performed by analysis algorithms such as MUMmer, but there is no limitation.

[0194] In this specification, the above-mentioned "high stringency conditions" refer to environmental conditions where non-specific hybridization is not likely to occur. Under high stringency conditions, hybrids can be formed with nucleic acids having target base sequences, but nucleic acids having non-specific base sequences are substantially unable to form hybrids. In general, high stringency conditions refer to conditions of low salt concentration and high temperature. Low salt concentration is, for example, 15 to 750 mM, preferably 15 to 500 mM, 15 to 300 mM or 15 to 200 mM. In addition, high temperature is, for example, 50 to 68°C or 55 to 70°C. As a specific example of high stringency conditions, conditions of washing with 65°C, 0.1×SSC and 0.1% SDS in washing after hybridization can be cited.

[0195] The proteins encoded by the above-mentioned tail fiber genes all have lytic activity against target bacteria.

[0196] (1-3) Genomic DNA

[0197] The first phage contains the above-mentioned fiber gene. There is no limitation on genes and base sequences other than the fiber gene. The phage genomic DNA can include, for example, genomic DNA comprising the following base sequences: the base sequence shown in any one of sequence numbers 8 to 10 (201015bp, 200185bp, and 200277bp, respectively); a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence in the region other than the tail fiber gene in the base sequence shown in any one of sequence numbers 8 to 10; and a base sequence in the region other than the tail fiber gene in the base sequence shown in any one of sequence numbers 8 to 10 having a sequence affinity of more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the base sequence shown in any one of sequence numbers 8 to 10. A base sequence having a sequence identity of at least 90.0%, at least 90.5%, at least 91.0%, at least 91.5%, at least 92.0%, at least 92.5%, at least 93.0%, at least 93.5%, at least 94.0 ... 5%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity.

[0198] It should be noted that, according to the software and analysis server used in this specification, as an index representing sequence identity, it is sometimes represented by Average Nucleotide Identity (ANI, average nucleotide consistency), etc., and they can also be used. It should be noted that for huge phage genome DNA, it is difficult to set the scope of matching configuration and comparison object as the whole scope. Therefore, the above sequence identity can be provided within the scope of automatic matching configuration by the above-mentioned software and Web service. For example, in the analysis using the BLAST server provided by NCBI, after automatically matching configuration within the maximum range that can be matched, the ratio of the scope as the comparison object in the whole range of phage genome DNA is calculated in the form of a value called Query Cover. In the case where a part of the range is matched and configured to calculate the sequence identity, the sequence identity of the base sequence in the whole range of phage genome DNA can also be estimated based on the result (sequence identity of the whole range). For example, the value of the sequence identity in the range that can be matched and compared (sequence identity of the matching range) can be multiplied by the Query Cover value and the value obtained as the estimated value of the sequence identity of the whole range. In this case, in order to improve the accuracy of the estimated value, further correction may be performed, for example, by taking into account the expected sequence identity in the range outside the matching range.

[0199] For example, when BLAST analysis was performed using GENETYX-NGS installed in GENETYX, the sequence identity (Average Nucleotide Identity: ANI) of sequence number 8 relative to sequence number 9 was 98% or more within a range of 96% or more of the entire length, and the sequence identity relative to sequence number 10 was 98% or more within a range of 95% or more of the entire length. Therefore, the sequence identity of the genomic DNA sequence of sequence number 8 relative to the entire length of the genomic DNA sequence of sequence number 9 can be estimated to be 94% or more. In addition, the sequence identity of the genomic DNA sequence of sequence number 8 relative to the entire length of the genomic DNA sequence of sequence number 10 can be estimated to be 93% or more.

[0200] It should be noted that when the genomic DNA of the bacteriophage in this specification is packaged, there are linear and circular situations. In addition, in the next generation genome sequencer analysis, after the genomic DNA is fragmented, the base sequence of each fragment is read, and they are connected, and the sequence is determined through such analysis. In the case of bacteriophages, most of the genomic DNA sequences used as references are not placed for connection (de novo assembly). Therefore, it is difficult to uniquely determine the starting point / end of the analyzed genome (Merrill, BD, et al. BMC Genomics, 201617, 679). The starting point / end of the genomic sequence to be compared can be different, which will be automatically considered in the analysis using software and analysis servers.

[0201] (1-4) Effect

[0202] The bacteriolytic agent of the present invention comprising the first bacteriophage can widely exhibit bacteriolytic activity against various species of Xanthomonas and can be applied to various plant diseases.

[0203] Generally speaking, when the specificity of a phage is too high, it cannot cover the diversity of target bacteria and the effect is limited. In addition, from an industrial point of view, it is preferably applicable to a variety of plant diseases. Therefore, the bacteriolytic agent of the present invention is very useful for a phage that exhibits lytic activity against a wide range of bacterial species.

[0204] <Second phage>

[0205] The second phage is characterized in that it contains a gene encoding a tail fiber protein containing a specific amino acid sequence in its genomic DNA and exhibits specific bacteriolytic activity against target bacteria.

[0206] (2-1) Tail fiber protein

[0207] The fiber protein comprises an amino acid sequence represented by SEQ ID NO: 11 consisting of 487 amino acid residues, or an amino acid sequence in which one amino acid other than positions 278 and 350 is substituted in the amino acid sequence represented by SEQ ID NO: 11. By comprising the amino acid sequence represented by SEQ ID NO: 11 or a mutant thereof, highly useful host specificity can be achieved, such as being specific to bacteria of a specific genus and exhibiting lytic activity against a wide range of bacterial species in the specific genus.

[0208] As described above, when one amino acid is substituted in the amino acid sequence shown in SEQ ID NO: 11, the position thereof may be any position other than position 278 and position 350, and is not particularly limited. For example, any one amino acid in the range of position 1 to position 250 in the amino acid sequence shown in SEQ ID NO: 11 may be substituted. Specifically, for example, one amino acid after position 50, after position 55, after position 60, after position 65, after position 66, after position 67, after position 68, after position 70, after position 80, after position 90, after position 100, after position 110, after position 120, after position 130, after position 140, after position 145, after position 150, after position 151, after position 152, after position 153, after position 154 in the amino acid sequence shown in SEQ ID NO: 11 may be substituted. Furthermore, for example, one amino acid at position 200 or earlier, position 190 or earlier, position 180 or earlier, position 170 or earlier, position 160 or earlier, position 159 or earlier, position 158 or earlier, position 157 or earlier, position 156 or earlier, position 155 or earlier, or position 154 or earlier in the amino acid sequence set forth in SEQ ID NO: 11 may be substituted. For example, one amino acid at position 50 to 200, position 67 to 156, position 80 to 156, position 100 to 156, position 110 to 156, position 120 to 156, position 130 to 156, position 140 to 156, position 150 to 156, position 153 to 156, position 154 to 156, or position 154 may be substituted. Since the amino acid region from positions 67 to 156 in the amino acid sequence shown in SEQ ID NO: 11 is considered to form a domain, when one amino acid is substituted, the amino acid may have the characteristics of the second phage regardless of its position as long as it is within the amino acid region.

[0209] The type of amino acid substitution is not particularly limited. For example, it can be a conservative substitution. In addition, for example, it can be a substitution within an amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr, Leu, Val, Ile, Val, Ala, Met, Pro) with a low polarity side chain. For example, it can be a substitution of an uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) or a neutral amino acid group (Asn, Gln, Thr, Ser, Tyr, Cys) with a hydrophilic side chain and a neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro). More specifically, for example, it can be a substitution of threonine (Thr) and alanine (Ala). As an example of an amino acid sequence after such substitution, the amino acid sequence shown in sequence number 45 can be cited. 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).

[0210] (2-2) Tail fiber gene

[0211] The second phage contains a fiber gene having a base sequence encoding the fiber protein in the genomic DNA of the phage.

[0212] The specific base sequence of the tail fiber gene is not particularly limited as long as it is the base sequence of the amino acid sequence shown in the above-mentioned sequence number 11 encoding or the amino acid sequence of 1 amino acid other than position 278 and position 350 replaced in the amino acid sequence shown in the sequence number 11. For example, it is the gene of the base sequence shown in the sequence number 12 comprising the amino acid sequence shown in the above-mentioned sequence number 11 encoding. In addition, for example, it is the gene of the base sequence shown in the sequence number 46 comprising the amino acid sequence shown in the above-mentioned sequence number 45 encoding.

[0213] The protein encoded by the fiber gene can achieve highly useful host specificity, which is specific to bacteria of a specific genus and exhibits bacteriolytic activity against a wide range of bacterial species in the specific genus.

[0214] (2-3) Genomic DNA

[0215] The second bacteriophage contains the above-mentioned tail fiber gene. There is no limitation on genes and base sequences other than the tail fiber gene. For example, the phage genomic DNA can include genomic DNA containing the following base sequences: the 63753bp base sequence shown in sequence number 13; the 63743bp base sequence shown in sequence number 47 or 48; the base sequence in the region other than the above-mentioned tail fiber gene in the base sequence shown in sequence number 13, 47 or 48 has one or more bases added, deleted and / or replaced; and the base sequence in the region other than the above-mentioned tail fiber gene in the base sequence shown in sequence number 13, 47 or 48 has 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 of the same base sequence as the base sequence shown in sequence number 13, 47 or 48. A base sequence having a sequence identity of 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more; a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in sequence number 13, 47, or 48; and a base sequence having a sequence identity of 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 in the base sequence shown in sequence number 13, 47, or 48, which hybridizes with a base sequence in a region other than the above-mentioned tail fiber gene under highly stringent conditions.

[0216] (2-4) Effect

[0217] The bacteriolytic agent of the present invention comprising the second bacteriophage can widely exhibit bacteriolytic activity against various species of Xanthomonas and can be applied to various plant diseases.

[0218] Generally speaking, when the specificity of a phage is too high, it cannot cover the diversity of target bacteria and the effect is limited. In addition, from an industrial point of view, it is preferably applicable to a variety of plant diseases. Therefore, the bacteriolytic agent of the present invention comprising the second phage is extremely useful for a phage that widely exhibits bacteriolytic activity against various bacterial species.

[0219] <The third phage>

[0220] The third phage is characterized by having a genomic DNA sequence containing a specific base sequence and exhibiting specific bacteriolytic activity against target bacteria.

[0221] (3-1) Genomic DNA

[0222] The genomic DNA sequence comprises or consists of the following base sequence: the 61291 bp base sequence shown in SEQ ID NO: 14; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in SEQ ID NO: 14; or a base sequence having a sequence identity of 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 to the base sequence shown in SEQ ID NO: 14. A bacteriophage having any genomic DNA sequence can also specifically adsorb to a target bacterium and inject its genomic DNA into the cells of the target bacterium.

[0223] (3-2) Effect

[0224] The bacteriolytic agent of the present invention comprising the third bacteriophage can exhibit bacteriolytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.

[0225] <The fourth bacteriophage>

[0226] The fourth bacteriophage is characterized in that it contains a gene encoding a tail fiber protein containing a specific amino acid sequence in its genomic DNA and exhibits specific bacteriolytic activity against target bacteria.

[0227] (4-1) Tail fiber protein

[0228] The tail fiber protein comprises: an amino acid sequence shown in sequence number 15 consisting of 604 amino acid residues, an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in sequence number 15, or an amino acid sequence having an amino acid sequence identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more with the amino acid sequence shown in sequence number 15. It is characterized in that any tail fiber protein has a recognition activity specific to a target bacterium.

[0229] (4-2) Tail fiber gene

[0230] The fourth bacteriophage contains a fiber gene having a base sequence encoding the fiber protein in the genomic DNA of the bacteriophage.

[0231] The specific base sequence of the tail fiber gene is, for example, a gene comprising the following base sequence: the base sequence shown in sequence number 16 encoding the amino acid sequence shown in sequence number 15 above, or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 16, and a base sequence having a base sequence identity of more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the base sequence shown in sequence number 16, or a base sequence that hybridizes with a base sequence complementary to the base sequence shown in sequence number 16 under high stringent conditions.

[0232] The proteins encoded by the above-mentioned tail fiber genes all have lytic activity against target bacteria

[0233] (4-3) Genomic DNA

[0234] The fourth phage contains the above-mentioned tail fiber gene. There is no limitation on genes and base sequences other than the tail fiber gene. For example, the phage genomic DNA can include genomic DNA containing the following base sequences: the 46393bp base sequence shown in sequence number 17; a base sequence in which one or more bases are added, deleted and / or replaced in the base sequence in the region other than the above-mentioned tail fiber gene in the base sequence shown in sequence number 3; and a base sequence in which the base sequence in the region other than the above-mentioned tail fiber gene in the base sequence shown in sequence number 17 has a sequence identity of more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the base sequence shown in sequence number 17; A base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown; 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, 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 matched with the base sequence shown in SEQ ID NO: 17.

[0235] (4-4) Effect

[0236] The bacteriolytic agent of the present invention comprising the fourth phage can exhibit bacteriolytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.

[0237] <Fifth Phage>

[0238] The fifth phage is characterized by having a genomic DNA sequence containing a specific base sequence, and the bacteriolytic agent of the present invention exhibits specific bacteriolytic activity against target bacteria.

[0239] (5-1) Genomic DNA

[0240] The DNA sequence of the genome of the phage of the fifth phage has the following base sequence: the base sequence of 43177 bp shown in sequence number 18 or the base sequence of 43741 bp shown in sequence number 19; or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 18 or 19; or a base sequence having a sequence identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more with the base sequence shown in sequence number 18 or 19; when matched with the base sequence shown in sequence number 18 or 19 A base sequence having 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 when configured; and a base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence shown in sequence number 18 or 19.

[0241] (5-2) Effect

[0242] The bacteriolytic agent of the present invention comprising the fifth phage can exhibit bacteriolytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.

[0243] <Sixth Phage>

[0244] The sixth phage is characterized in that it contains a gene encoding a protein newly discovered in the present invention (sometimes referred to simply as "the protein of the present invention" in this specification) in its genomic DNA and exhibits specific bacteriolytic activity against target bacteria.

[0245] (6-1) Protein of the present invention

[0246] The protein of the present invention comprises: an amino acid sequence represented by SEQ ID NO: 21 consisting of 84 amino acid residues; an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence represented by SEQ ID NO: 21; or an amino acid sequence having an amino acid sequence identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more to the amino acid sequence represented by SEQ ID NO: 21. The protein of the present invention also has target bacteria-specific recognition activity.

[0247] The protein of the present invention is a novel protein that shows an important role in determining the host range.

[0248] (6-2) Gene encoding the protein of the present invention

[0249] This sixth phage contains a gene encoding the above-mentioned protein of the present invention (sometimes simply referred to as "the gene of the present invention" in the present specification) in the genomic DNA of the phage.

[0250] The gene of the present invention is not particularly limited as long as it is a base sequence encoding the amino acid sequence shown in the above sequence number 21. As a specific base sequence, for example, it is a gene comprising the following base sequence: the base sequence shown in sequence number 22 encoding the amino acid sequence shown in sequence number 21; or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 22; and a base sequence having a base sequence identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the base sequence shown in sequence number 22; or a base sequence that hybridizes with a base sequence complementary to the base sequence shown in sequence number 22 under highly stringent conditions.

[0251] The proteins encoded by the genes of the present invention mentioned above all have bacteriolytic activity against target bacteria.

[0252] (6-3) Genomic DNA

[0253] The sixth phage contains the gene of the present invention described above. There is no limitation on genes and base sequences other than the gene of the present invention. For example, the phage genomic DNA can include genomic DNA containing the following base sequences: a base sequence of 42708 bp shown in sequence number 23; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence in the region other than the gene of the present invention described above in the base sequence shown in sequence number 23; and a base sequence in which the base sequence in the region other than the gene of the present invention described above has a sequence identity of 95% or more, 96% or more, 97% or more, 98% or more or 99% or more with the base sequence shown in sequence number 23; A base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in SEQ ID NO: 23; and a base sequence having 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 when matched with the base sequence shown in SEQ ID NO: 23.

[0254] (6-4) Effect

[0255] The bacteriolytic agent of the present invention comprising the sixth phage can exhibit bacteriolytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.

[0256] <7th Phage>

[0257] The seventh phage is characterized in that it contains a gene encoding a tail tube protein containing a specific amino acid sequence in its genomic DNA and exhibits specific bacteriolytic activity against target bacteria.

[0258] (7-1) Tail tube protein

[0259] The tail tube protein includes tail tube protein A and tail tube protein B. The tail tube protein A includes: an amino acid sequence represented by sequence number 24 or 49 consisting of 205 amino acid residues; an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence represented by sequence number 24 or 49; or an amino acid sequence having an amino acid sequence identity of 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more with the amino acid sequence represented by sequence number 24 or 49.

[0260] The amino acid substitution here may include amino acid substitution at one or more positions selected from the group consisting of position 28, position 108, position 110, position 153, position 157, position 189 and position 201 in the amino acid sequence set forth in SEQ ID NO: 24 or 49. For example, substitution at two or more, three or more, four or more, five or more, six or more, or all of these positions may be included.

[0261] The specific type of amino acid substitution is not particularly limited. For example, it can be a conservative substitution, or it can include one or more non-conservative substitutions. Specifically, for example, the amino acid sequence of the tail tube protein A of the 7th bacteriophage can be the amino acid sequence shown in sequence number 60. The amino acid sequence is identical to the amino acid sequence shown in sequence number 24 or 49 except the amino acid at the position listed below: the 28th is glutamic acid (Glu) or aspartic acid (Asp), the 108th is serine (Ser) or asparagine (Asn), the 110th is glutamine (Gln) or histidine (His), the 153rd is glutamine (Gln) or lysine (Lys), the 157th is aspartic acid (Asp) or asparagine (Asn), the 189th is tyrosine (Tyr) or phenylalanine (Phe), and the 201st is valine (Val) or tyrosine (Tyr).

[0262] The above-mentioned tail tube protein B comprises: the amino acid sequence shown in sequence number 25 (to be precise, sequence number 57) or 50 composed of 834 amino acid residues; an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in sequence number 25 (to be precise, sequence number 57) or 50; or an amino acid sequence having an amino acid sequence identity of more than 97%, more than 97.5%, more than 97.6%, more than 98%, more than 98.5%, more than 99% or more than 99.5% with the amino acid sequence shown in sequence number 25 (to be precise, sequence number 57) or 50.

[0263] The amino acid substitution herein may include amino acid substitution at one or more positions selected from the group consisting of position 25, position 53, position 216, position 221, position 272, position 389, position 395, position 410, position 425, position 472, position 607, position 623, position 662, position 670, position 699, position 707, position 757, position 763, position 764, and position 765 in the amino acid sequence set forth in SEQ ID NO: 57 or 50. For example, substitution at two or more, three or more, four or more, five or more, ten or more, fifteen or more, or all of these positions may be included.

[0264] The specific type of amino acid substitution is not particularly limited. For example, it can be a conservative substitution, or it can contain one or more non-conservative substitutions. Specifically, for example, the amino acid sequence of the tail tube protein B of the 7th phage can be the amino acid sequence shown in sequence number 61. The amino acid sequence is the same as the amino acid sequence shown in sequence number 57 or 50, except for the amino acids at the positions listed below: the 25th position is alanine (Ala) or proline (Pro), the 53rd position is alanine (Ala) or serine (Ser), the 216th position is alanine (Ala) or proline (Pro), the 221st position is histidine (His) or tyrosine (Tyr), the 272nd position is valine (Val) or (Ile), the 389th position is alanine (Ala) or glutamic acid (Glu), the 40th position is succinylcholine (Ser), the 41st position is glutamic acid (Glu), the 42nd position is glutamic acid (Glu), the 43rd position is succinylcholine (Ser), the 44th position is glutamic acid (Glu), the 45th position is glutamic acid (Glu), the 46th position is glutamic acid (Glu), the 47th position is glutamic acid (Glu), the 48th position is glutamic acid (Glu), the 49th position is glutamic acid (Glu), the 54th position is glutamic acid (Glu), the 55th position is glutamic acid (Glu), the 56th position is glutamic acid (Glu), the 57th position is glutamic acid (Glu), the 58th position is glutamic acid (Glu), the 59th position is glutamic acid (Glu), the 61st position is glutamic acid (Glu), the 62nd position is glutam The 395th position is serine (Ser) or alanine (Ala), the 410th position is valine (Val) or isoleucine (Ile), the 425th position is isoleucine (Ile) or valine (Val), the 472nd position is glycine (Gly) or alanine (Ala), the 607th position is alanine (Ala) or serine (Ser), the 623rd position is isoleucine (Ile) or valine (Val), the 662nd position is arginine (Arg) or serine (Ser), the 670th position is leucine (Le u) or glutamine (Gln), threonine (Thr) or asparagine (Asn) at position 699, aspartic acid (Asp) or glycine (Gly) at position 707, serine (Ser) or alanine (Ala) at position 757, glycine (Gly) or serine (Ser) at position 763, serine (Ser) or asparagine (Asn) at position 764, and methionine (Met) or valine (Val) at position 765.

[0265] Any tail tube protein is associated with a recognition activity specific to a target bacterium.

[0266] (7-2) Tail tube gene

[0267] The seventh phage contains a tail tube gene having a base sequence encoding the tail tube protein in the genomic DNA of the phage.

[0268] The tail tube gene is not particularly limited as long as it is a gene encoding a tail tube protein, and includes a tail tube protein A gene and a tail tube protein B gene.

[0269] The specific base sequence of the tail tube protein A gene is, for example, a gene comprising the following base sequence: the base sequence shown in SEQ ID NO: 26 or 51 encoding the amino acid sequence shown in SEQ ID NO: 24 or 49; or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in SEQ ID NO: 26 or 51; and a base sequence having a base sequence identity of 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more with the base sequence shown in SEQ ID NO: 26 or 51; or a base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 26 or 51. The base sequence of the tail tube protein A gene may be a base sequence encoding the amino acid sequence shown in SEQ ID NO: 60.

[0270] A specific base sequence of the caudal tube protein B gene is, for example, a gene comprising the following base sequence: a base sequence as shown in SEQ ID NO: 27 (to be precise, SEQ ID NO: 58) or 52 encoding the amino acid sequence as shown in SEQ ID NO: 25 (to be precise, SEQ ID NO: 57) or 50; or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in SEQ ID NO: 27 (to be precise, SEQ ID NO: 58) or 52; and a base sequence having a base sequence identity of 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more with the base sequence as shown in SEQ ID NO: 27 (to be precise, SEQ ID NO: 58) or 52; or a base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence as shown in SEQ ID NO: 27 (to be precise, SEQ ID NO: 58) or 52. The base sequence of the caudal tube protein B gene may be a base sequence encoding the amino acid sequence as shown in SEQ ID NO: 61.

[0271] The proteins encoded by the above tail tube genes all have bacteriolytic activity against target bacteria.

[0272] (7-3) Genomic DNA

[0273] The seventh phage contains the above-mentioned tail tube gene. There is no limitation on genes and base sequences other than the tail tube gene. For example, the phage genomic DNA can include genomic DNA containing the following base sequences: the 44716bp base sequence shown in sequence number 28; the 44829bp base sequence shown in sequence number 29 or the 44585bp base sequence shown in sequence number 53; or the base sequence in the region other than the above-mentioned tail tube gene in the base sequence shown in sequence number 28, 29 or 53 is added, deleted and / or substituted with one or more bases; and the base sequence in the region other than the above-mentioned tail tube gene in the base sequence shown in sequence number 28, 29 or 53 has 95% or more, 96% or more, 97% or more, 98% or more or 99% or more similarity with the base sequence shown in sequence number 28, 29 or 53. A base sequence having a sequence identity of % or more; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in SEQ ID NO: 28, 29 or 53; and a base sequence having 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 when matched with the base sequence shown in SEQ ID NO: 28, 29 or 53.

[0274] (7-4) Effect

[0275] The bacteriolytic agent of the present invention comprising the seventh bacteriophage can widely exhibit bacteriolytic activity against various species of the genus Xanthomonas and can be applied to various plant diseases.

[0276] Generally speaking, when the specificity of a phage is too high, it cannot cover the diversity of target bacteria and the effect is limited. In addition, from an industrial point of view, it is preferably applicable to a variety of plant diseases. Therefore, a phage that exhibits lytic activity against two or more bacterial species, such as the bacteriolytic agent of the present invention containing the seventh phage, is extremely useful.

[0277] <Bacteriophage No. 8>

[0278] The eighth phage is characterized in that it has a genomic DNA sequence containing a specific base sequence and exhibits specific bacteriolytic activity against target bacteria.

[0279] (8-1) Genomic DNA

[0280] As the above-mentioned genomic DNA sequence, there can be mentioned a genomic DNA sequence comprising the following base sequences or consisting of the following base sequences: a base sequence shown in any one of sequence numbers 32 to 36 (44388bp, 44377bp, 45279bp, 44378bp, 44408bp, respectively); a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 32 to 36; a base sequence having a base sequence of 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, or 100% or more when matched with the base sequence shown in any one of sequence numbers 32 to 36. 89% or more, 90% 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 sequence identity.

[0281] (8-2) Effect

[0282] The bacteriolytic agent of the present invention comprising the 8th phage can show bacteriolytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.

[0283] <Phage No. 9>

[0284] The ninth phage is characterized in that it contains a gene encoding a tail tube protein having a specific amino acid sequence in its genomic DNA and exhibits specific bacteriolytic activity against target bacteria.

[0285] (9-1) Tail tube protein

[0286] The tail tube protein includes tail tube protein A and tail tube protein B. The tail tube protein A includes: an amino acid sequence shown in sequence number 37 consisting of 206 amino acid residues; an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in sequence number 37; or an amino acid sequence having an amino acid sequence identity of 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 with the amino acid sequence shown in sequence number 37.

[0287] The above-mentioned tail tube protein B comprises: the amino acid sequence shown in sequence number 38 or 54 consisting of 847 amino acid residues; an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in sequence number 38 or 54; or an amino acid sequence having a sequence identity of more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 97.5%, more than 98%, more than 98.5%, more than 99%, more than 99.1%, more than 99.2%, more than 99.3%, more than 99.4% or more of amino acids with the amino acid sequence shown in sequence number 38 or 54.

[0288] The amino acid substitution here may include amino acid substitution at one or more positions selected from position 61, position 108, position 404, position 679, position 682 and position 727 in the amino acid sequence shown in SEQ ID NO: 38 or 54. For example, substitution at two or more, three or more, four or more, five or more, or all of these positions may be included.

[0289] The specific type of amino acid substitution is not particularly limited. For example, it can be a conservative substitution, or it can include one or more non-conservative substitutions. For example, it can be a substitution within an amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr, Leu, Val, Ile, Val, Ala, Met, Pro) with a low polarity branched chain. For example, it can be a substitution of an uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) or a neutral amino acid group (Asn, Gln, Thr, Ser, Tyr, Cys) with a hydrophilic branched chain and a neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro). More specifically, for example, it can be a substitution of threonine (Thr) with alanine (Ala).

[0290] As a specific amino acid sequence, there can be mentioned the amino acid sequence shown in SEQ ID NO: 59. This amino acid sequence is the same as the amino acid sequence shown in SEQ ID NO: 38 except for the amino acids at the positions listed below: 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).

[0291] A phage containing the following amino acid sequence is also included in the 9th phage of the present invention: the amino acid sequence of the tail tube protein B is 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: 59, 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 amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 59.

[0292] Any tail tube protein is associated with a recognition activity specific to a target bacterium.

[0293] (9-2) Tail tube gene

[0294] The ninth phage contains a tail tube gene containing a base sequence encoding the above-mentioned tail tube protein in the genomic DNA of the phage.

[0295] The tail tube gene is not particularly limited as long as it is a gene encoding a tail tube protein, and includes a tail tube protein A gene and a tail tube protein B gene.

[0296] The specific base sequence of the tail tube protein A gene is, for example, a gene comprising the following base sequence: the base sequence shown in SEQ ID NO: 39 encoding the amino acid sequence shown in SEQ ID NO: 37; or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in SEQ ID NO: 39; or a base sequence having a base sequence identity of 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 with the base sequence shown in SEQ ID NO: 39; or a base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 39. The base sequence of the tail tube protein A gene may be a base sequence encoding the amino acid sequence shown in SEQ ID NO: 59.

[0297] Specific base sequences of the tail tube protein B gene include, for example, a gene comprising the following base sequences: a base sequence as shown in SEQ ID NO: 40 or 55 encoding the amino acid sequence as shown in SEQ ID NO: 38 or 54; or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence as shown in SEQ ID NO: 40 or 55; or a base sequence having a base sequence identity of 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 with the base sequence as shown in SEQ ID NO: 40 or 55; or a base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence as shown in SEQ ID NO: 40 or 55. Specific base sequences of the tail tube protein B gene include, for example, a base sequence encoding the amino acid sequence as shown in SEQ ID NO: 59.

[0298] The proteins encoded by the above tail tube genes all have bacteriolytic activity against target bacteria.

[0299] (9-3) Genomic DNA

[0300] The 9th phage contains the above-mentioned tail tube gene. There is no limitation on genes and base sequences other than the tail tube gene. For example, the phage genomic DNA can include genomic DNA containing the following base sequences: the base sequence of 43667 bp shown in sequence number 41 or the base sequence of 43336 bp shown in sequence number 56; or the base sequence in the region other than the above-mentioned tail tube gene in the base sequence shown in sequence number 41 or 56 is added, deleted and / or substituted with one or more bases; and the base sequence in the region other than the above-mentioned tail tube gene in the base sequence shown in sequence number 41 or 56 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 of the base sequence shown in sequence number 41 or 56. A base sequence having a sequence identity of 90% or more, 97% or more, 98% or more or 99% or more to the base sequence set forth in SEQ ID NO: 41 or 56; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence set forth in SEQ ID NO: 41 or 56; and a base sequence having a sequence identity of 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 when matched with the base sequence set forth in SEQ ID NO: 41 or 56.

[0301] (9-4) Effect

[0302] The bacteriolytic agent of the present invention can widely exhibit bacteriolytic activity against various species of Xanthomonas and can be applied to various plant diseases.

[0303] Generally speaking, when the specificity of a phage is too high, it cannot cover the diversity of target bacteria and the effect is limited. In addition, from an industrial point of view, it is preferably applicable to a variety of plant diseases. Therefore, a phage that exhibits lytic activity against two or more bacterial species, such as the bacteriolytic agent of the present invention, is extremely useful.

[0304] <10th Phage>

[0305] The tenth phage is characterized in that it contains a gene encoding a tail fiber protein containing a specific amino acid sequence in its genomic DNA and exhibits specific bacteriolytic activity against target bacteria.

[0306] (10-1) Tail fiber protein

[0307] The tail fiber protein comprises: an amino acid sequence shown in sequence number 42 consisting of 568 amino acid residues; an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in sequence number 42; or an amino acid sequence having an amino acid sequence identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in sequence number 42. Any tail fiber protein has target bacteria-specific recognition activity.

[0308] (10-2) Tail fiber gene

[0309] The tenth bacteriophage contains a fiber gene having a base sequence encoding the fiber protein in the genomic DNA of the bacteriophage.

[0310] The specific base sequence of the tail fiber gene is, for example, a gene comprising the following base sequence: the base sequence shown in sequence number 43 encoding the amino acid sequence shown in sequence number 42 above; or a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 43; and a base sequence having a sequence identity of more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of bases with the base sequence shown in sequence number 43; or a base sequence that hybridizes with a base sequence complementary to the base sequence shown in sequence number 43 under highly stringent conditions.

[0311] The proteins encoded by the above-mentioned tail fiber genes all have lytic activity against target bacteria.

[0312] (10-3) Genomic DNA

[0313] The 10th phage contains the above-mentioned tail fiber gene. There is no limitation on genes and base sequences other than the tail fiber gene. For example, the phage genomic DNA can include genomic DNA containing the following base sequences: the 76340 bp base sequence shown in sequence number 44; a base sequence in which one or more bases are added, deleted and / or replaced in the base sequence in the region other than the above-mentioned tail fiber gene in the base sequence shown in sequence number 44; and a base sequence in which the base sequence in the region other than the above-mentioned tail fiber gene in the base sequence shown in sequence number 44 has a sequence identity of more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the base sequence shown in sequence number 44; 4; 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, 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 matched with the base sequence shown in SEQ ID NO:44.

[0314] (10-4) Effect

[0315] The bacteriolytic agent of the present invention can widely exhibit bacteriolytic activity against various species of Xanthomonas and can be applied to various plant diseases.

[0316] 2. Plant disease control composition

[0317] 2-1. Overview

[0318] A second aspect of the present invention is a composition, particularly a composition that can be used for controlling plant diseases. The composition of the present invention is characterized by comprising the bacteriolytic agent described in the first aspect as an active ingredient.

[0319] The composition of the present invention can be used for plant disease control purposes to provide a sustainable pesticide for bacterial plant diseases that can specifically prevent or treat target plant diseases while being safe for humans and having no phytotoxicity to the environment.

[0320] In addition, in this specification, when it expresses "plant disease control composition", it means the case where the composition of this invention is used for plant disease control.

[0321] 2-2. Composition

[0322] 2-2-1. Composition

[0323] The composition of the present invention includes, as an essential component, the bacteriolytic agent described in the first embodiment, i.e., a bacteriophage, as an effective component. In addition, agronomically acceptable carriers and / or media may be included within the range that the bacteriolytic activity of the target bacteria of the bacteriophage is not impaired or inhibited. In addition, other effective components may be included as needed. The following specifically describes each component.

[0324] (1) Active ingredient (bacteriolytic agent)

[0325] The composition of the present invention contains the bacteriolytic agent described in the first embodiment as an essential active ingredient. In the plant disease control composition, the target bacteria of the present invention can be lysed by the active ingredient, and thus plant diseases caused by the target bacteria can be prevented or treated.

[0326] The specific structure of the bacteriolytic agent has been described in detail in the first embodiment, and therefore the description here is omitted.

[0327] When used for plant disease control purposes, the amount of the active ingredient contained per unit amount in the composition is determined by various conditions such as the dosage form, the type of plant pathogenic bacteria, the type of target plant, the application site, and the application method. For the bacteriophage as an active ingredient, it is preferably contained in an amount sufficient to contact and infect the plant pathogenic bacteria that infect the target plant. Therefore, within the scope of technical common sense in the field, it is sufficient to consider and determine each condition in such a way that the bacteriolytic agent contained in the plant disease control composition of the present invention reaches an effective amount for the target bacteria after application.

[0328] The plant disease control composition of the present invention can specifically recognize the Xanthomonas bacteria specifically exemplified below, and can contain one or more other phages having bacteriolytic activity as active ingredients. For example, even if the target bacteria are the same, as long as the phages recognize different cell surface receptors, a synergistic effect or complementary effect of bacteriolytic activity can be expected through their combination.

[0329] When the composition of the present invention includes multiple phages, the specific type of phage is not particularly limited. For example, only the phages described in this specification may be included as active ingredients, or other arbitrary phages may be additionally included as active ingredients. In addition, phages with similar host ranges and / or phages with dissimilar host ranges may be included. As a combination of phages with similar host ranges, for example: a combination that shows lytic activity against bacteria of the same genus (e.g., Xanthomonas), a combination of phages that show lytic activity against bacteria of more than one species of the same genus (e.g., Xanthomonas sarboricola), a combination of phages that show lytic activity against bacteria of more than one pathotype of the same genus (e.g., Xanthomonas arboricola pv. pruni), etc. In addition, examples of combinations of phages with dissimilar host ranges include: a combination of a phage that exhibits lytic activity against bacteria belonging to a specific genus (e.g., Xanthomonas) and a phage that does not exhibit lytic activity, a combination of a phage that exhibits lytic activity against bacteria of a specific pathotype of a specific genus (e.g., Xanthomonas arboricola pv. pruni) and a phage that does not exhibit lytic activity, a combination of a phage that exhibits lytic activity against bacteria of a specific species of a specific genus (e.g., Xanthomonas arboricola) and a phage that does not exhibit lytic activity, a combination of a phage that specifically exhibits lytic activity against bacteria of a specific genus (e.g., Xanthomonas arboricola), species (e.g., Xanthomonas arboricola), and pathotype (e.g., Xanthomonas arboricola pv. pruni) and a phage that nonspecifically exhibits lytic activity, and the like.

[0330] For example, the composition of the present invention may contain two or more phages from the first to tenth phages described in this specification in combination. Specifically, for example, two or more phages selected from the first phage, the second phage, the seventh phage, the ninth phage, and the tenth phage may be contained in combination. In addition, for example, two or more phages selected from the third phage, the fourth phage, the fifth phage, the sixth phage, and the eighth phage may be contained in combination. In addition, for example, one or more phages selected from the first phage, the second phage, the seventh phage, the ninth phage, and the tenth phage and one or more phages selected from the third phage, the fourth phage, the fifth phage, the sixth phage, and the eighth phage may be contained in combination.

[0331] The number of the first to tenth phages recorded in this specification included in the composition of the present invention is not particularly limited as long as it is one or more. For example, it may include 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. In addition, the composition of the present invention may include a plurality of phages whose genomic DNA sequences are different from each other, although they are included in the range of each phage (e.g., the second phage, etc.). Specifically, it may include two or more second phages and / or two or more seventh phages.

[0332] For example, a phage that contains a gene encoding the amino acid sequence shown in sequence number 1 as a common amino acid sequence for tail fiber proteins in its genomic DNA may specifically include: a phage that contains a gene encoding the amino acid sequence shown in any one of sequence numbers 2 to 4 in its genomic DNA; and an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in any one of sequence numbers 1 to 4; or a phage that contains a gene encoding an amino acid sequence with a sequence identity of 90% or more in its genomic DNA. Specific examples of genes encoding such amino acid sequences include: a gene containing a base sequence shown in any one of sequence numbers 5 to 7; and a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 5 to 7, or a base sequence with a sequence identity of 90% or more thereto. As the genomic DNA of a bacteriophage containing such a gene, for example, a genomic DNA containing the following base sequences can be listed: a genomic DNA containing the base sequence shown in any one of sequence numbers 8 to 10; and a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 8 to 10; a base sequence having a sequence identity of 90% or more with the base sequence shown in any one of sequence numbers 8 to 10; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 8 to 10 other than the base sequence encoding the amino acid sequence shown in any one of sequence numbers 2 to 4; or a base sequence having a sequence identity of 80% or more with a nucleotide sequence in the base sequence shown in any one of sequence numbers 8 to 10 other than the base sequence encoding the amino acid sequence shown in any one of sequence numbers 2 to 4.

[0333] In addition, examples include: a phage containing a gene encoding the amino acid sequence shown in sequence number 11 or 45 as the amino acid sequence of the tail fiber protein in its genomic DNA; and a phage containing a gene encoding an amino acid sequence in which one or more amino acids are added, deleted and / or substituted at positions other than 278 and 350 in the amino acid sequence shown in sequence number 11, or an amino acid sequence having a sequence identity of 90% or more thereto. Specific examples of genes encoding such amino acid sequences include: a gene containing the base sequence shown in sequence number 12 or 46; and a base sequence in which one or more bases are added, deleted and / or substituted at positions other than 278 and 350 in the base sequence shown in sequence number 12 or 46, or a base sequence having a sequence identity of 90% or more thereto. In addition, as specific genomic DNAs of bacteriophages containing such genes, for example, genomic DNAs containing the base sequences shown in sequence numbers 13, 47 or 48, and genomic DNAs containing the following base sequences: a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence numbers 13, 47 or 48; a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence numbers 13, 47 or 48; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence other than the base sequence encoding the amino acid sequence shown in sequence numbers 11 or 45 in the base sequence shown in sequence numbers 13, 47 or 48; or a base sequence having a sequence identity of 80% or more with the base sequence other than the base sequence encoding the amino acid sequence shown in sequence numbers 11 or 45 in the base sequence shown in sequence numbers 13, 47 or 48.

[0334] In addition, the genomic DNA of the bacteriophage can be listed, for example: a genomic DNA comprising a base sequence shown in any one of sequence numbers 14, 18, 19 and 32 to 36; and a genomic DNA comprising a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 14, 18, 19 and 32 to 36, and a base sequence having a sequence identity of more than 90%.

[0335] In addition, examples include: a phage containing a gene encoding the amino acid sequence shown in sequence number 15 or 42 as the amino acid sequence of the tail fiber protein in its genomic DNA; and a phage containing a gene encoding an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in sequence number 15 or 42, or an amino acid sequence having a sequence identity of 90% or more thereto in its genomic DNA. Specific examples of genes encoding such amino acid sequences include: a gene containing the base sequence shown in sequence number 16 or 43; and a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 16 or 43, or a base sequence having a sequence identity of 90% or more thereto. In addition, as specific genomic DNAs of bacteriophages containing such genes, for example, genomic DNAs containing the base sequence shown in sequence numbers 17 or 44, and genomic DNAs containing the following base sequences can be listed: a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence numbers 17 or 44; a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence numbers 17 or 44; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence other than the base sequence encoding the amino acid sequence shown in sequence numbers 15 or 42 in the base sequence shown in sequence numbers 17 or 44; or a base sequence having a sequence identity of 80% or more with the base sequence other than the base sequence encoding the amino acid sequence shown in sequence numbers 15 or 42 in the base sequence shown in sequence numbers 17 or 44.

[0336] In addition, examples include: a phage containing a gene encoding the amino acid sequence shown in SEQ ID NO: 21 in its genomic DNA; and a phage containing a gene encoding an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having a sequence identity of 90% or more thereto in its genomic DNA. Specific examples of genes encoding such amino acid sequences include: a gene containing the base sequence shown in SEQ ID NO: 22; and a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in SEQ ID NO: 22, or a base sequence having a sequence identity of 90% or more thereto. In addition, as specific genomic DNAs of bacteriophages containing such genes, for example, genomic DNAs containing the base sequence shown in sequence number 23, and genomic DNAs containing the following base sequences can be listed: a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in sequence number 23; a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence number 23; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence other than the base sequence encoding the amino acid sequence shown in sequence number 21 in the base sequence shown in sequence number 23; or a base sequence having a sequence identity of 80% or more with the base sequence other than the base sequence encoding the amino acid sequence shown in sequence number 21 in the base sequence shown in sequence number 23.

[0337] In addition, for example, there can be mentioned a phage comprising in its genomic DNA a TTPA (Tail-Tubular protein A) gene comprising the amino acid sequence shown in SEQ ID NO: 24, 49, 60 or 37 and a TT PB (Tail-Tubular protein B) gene comprising the amino acid sequence shown in SEQ ID NO: 25 (to be precise, SEQ ID NO: 57), 50, 61 or 38, 54 or 59; and a phage comprising in its genomic DNA a TTPA gene encoding an amino acid sequence in which one or more amino acids are added to, deleted from and / or substituted in the amino acid sequence shown in SEQ ID NO: 24, 49 or 37, or 25 (to be precise, SEQ ID NO: 57), 50 or 38, 54 or 59, or an amino acid sequence having 90% or more sequence identity thereto. Specific examples of genes encoding such amino acid sequences include: genes comprising the base sequences shown in sequence numbers 26 or 39 or 27 (to be precise, sequence number 58), 40 or 55; and base sequences in which one or more bases are added, deleted and / or substituted in the base sequences shown in sequence numbers 26 or 39, or 27 (to be precise, sequence number 58), 40 or 55, or base sequences having a sequence identity of 90% or more thereto. In addition, as specific genomic DNAs of bacteriophages containing such genes, for example, genomic DNAs containing the base sequence shown in any one of sequence numbers 28, 29, 41 or 56, and genomic DNAs containing the following base sequences: a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 28, 29, 41 or 56; a base sequence having a sequence identity of 90% or more with the base sequence shown in any one of sequence numbers 28, 29, 41 or 56; a base sequence in which one or more bases are added, deleted and / or substituted in the base sequence shown in any one of sequence numbers 28, 29, 41 or 56; A base sequence in which one or more bases are added, deleted and / or substituted in a base sequence other than a base sequence encoding an amino acid sequence represented by sequence numbers 24 or 37, or 25 (to be precise, sequence number 57), 50 or 38, 54 or 59 in a base sequence shown in one of the above; or a base sequence having a sequence identity of 80% or more with a base sequence other than a base sequence encoding an amino acid sequence represented by sequence numbers 24 or 37, or 25 (to be precise, sequence number 57), 50 or 38, 54 or 59 in a base sequence shown in any of the above.

[0338] The sequence identity in this specification is not particularly limited. Specifically, for example, relative to the sequence used as a reference, it can be 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, 94.6% or more, 95.7% or more, 96.8% or more, 97.9% or more, 98.1% or more, 99.2% or more, 100. .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.

[0339] (2) Agronomically acceptable carriers and media

[0340] "Agriculturally acceptable carriers and / or media" refer to substances that facilitate the application of the composition, can maintain the viability and infectivity of the bacteriophage as the active ingredient, and / or can control the rate of action, and the field application of the substance causes little or no harmful effects on the environment such as soil and water quality, and thus has little or no harmfulness to animals, especially humans.

[0341] (2-1) Carrier

[0342] As specific examples of agronomically acceptable carriers, surfactants, protective agents, and excipients can be cited. In addition, if desired, a small amount of wetting agents, emulsifiers, and pH buffers can also be used. The carrier can be pre-matched or can be matched just before application.

[0343] Surfactants have the effect of improving the physical and chemical properties of the composition such as wettability, emulsification, dispersibility, penetration, adhesion, defoaming and expansibility on the plant body. Surfactants can be used as the main component of pesticide adjuvants called spreaders. Examples of spreaders include: nonionic surfactants, combinations of nonionic surfactants and anionic surfactants, paraffins, and polyoxyethylene resin acid esters. More specifically, examples include: polyoxyethylene alkyl ether compounds, polyoxyethylene fatty acid ester compounds, lignin sulfonate compounds, naphthyl methanesulfonate compounds, alkyl sulfosuccinate compounds, and tetraalkylammonium salt compounds.

[0344] In the case of bacteriophages, the protective agent can be expected to have the effect of reducing damage caused by ultraviolet rays, etc. Examples thereof include skim milk, caseins, gelatins, and the like.

[0345] Examples of the excipient include glucose, lactose, sucrose, gelatin, starch, malt, and wheat flour.

[0346] (2-2) Solvent

[0347] Specific examples of the agronomically acceptable solvent include water (including aqueous solutions), buffer solutions, and liquid culture media. The solvent is preferably a sterile liquid.

[0348] (3) Other active ingredients

[0349] The composition of the present invention may contain, in addition to the bacteriolytic agent described in the first embodiment, one or more other active ingredients having the same and / or different pharmacological actions within the range that does not affect the bacteriolytic activity of the bacteriophage constituting the bacteriolytic agent.

[0350] The types of other active ingredients are not limited. For example, they may be bacteriophages that have lytic activity against the same and / or different bacteria. Examples of bacteriophages that have lytic activity against the same bacteria include other bacteriophages that specifically recognize and bind to Xanthomonas bacteria, similarly to the bacteriophages that constitute the bacteriolytic agent described in the first embodiment.

[0351] Furthermore, insecticides, herbicides, fertilizers (eg, urea, ammonium nitrate, superphosphate), and known chemical pesticides, antibiotics, and biopesticides may be contained as other active ingredients.

[0352] 2-2-2. Dosage form

[0353] When used as a plant disease control composition, the dosage form of the composition of the present invention can be any dosage form as long as it can maintain the infection site of the target bacteria on the target plant, the adhesion of the target plant, and / or the ease of infection of the bacteriophage as an active ingredient to the target bacteria. For example, it can be made into a liquid preparation or a water-dispersible agent in a liquid state formed by suspending the plant disease control composition in an appropriate solution, or it can be made into a solid state powder, granules, or gel that is mixed with a carrier and solidified. For example, when the infection site of the target bacteria in the target plant is the leaves, flowers, fruits, stems, branches or trunks of the aboveground part, it is not limited, and it is preferably a liquid preparation, water dispersant or gel that is widely distributed over these infection sites and has high fixation. On the other hand, when the infection site of the target bacteria is the root or underground stem of the underground part, it is not limited, and it is preferably a powder or granule that is slowly released in the soil and can continuously exert an effect at the infection site.

[0354] 2-3. Application method

[0355] When used as a plant disease control composition, the method of applying the composition of the present invention can be any method that can apply the plant disease control composition to the target plant, and methods known in the art can be used without particular limitation. From the aspect of being able to invade the entire surface of the plant body such as the stems and leaves and roots of the target plant, the bacteriophage as the active ingredient of the plant disease control composition can be applied by an appropriate method corresponding to the target. For example, when the application site is an aboveground part such as the stems and leaves, the plant disease control composition can be applied in a manner that the plant disease control composition is in direct contact with the application site. Direct contact can include, for example, coating, spraying, spreading or dipping of the plant disease control composition at the application site. It is particularly preferred that the application be carried out on the infected site of the target plant infected by the target bacteria, or on the site where there is a hidden danger of infection. In addition, when the application site is an underground part such as the root, it can be added to the soil, or in the case of a culture medium, it can be added to the culture medium, which can be indirect. The so-called "soil" here is not particularly limited as long as it is soil in which the target plant can grow. Cultivation soil containing appropriate nutrients and having an appropriate pH value can usually be used. There is no restriction on the site of the soil. In addition, "culture medium" refers to a culture medium for cultivating the target plant artificially prepared. It can be a solid culture medium such as an agar culture medium, or a liquid culture medium. Examples of culture medium include, for example, an isolation bed, a root zone restriction pot, or a seedling bed. The composition of the culture medium can be a culture medium composition known in the art. It can be appropriately selected according to the type of plant, etc.

[0356] 2-4. Target plants

[0357] The target plant of the plant disease control composition of the present invention is a plant that may develop a plant disease caused by the target bacteria of the present invention, and its species is not particularly limited. It can be any plant in angiosperms or gymnosperms. In addition, it can be either a herbaceous plant or a woody plant. As a preferred specific example of the target plant, important agricultural plants, such as crop plants such as cereals, vegetables, and fruits, and flower plants can be cited. Specifically, in monocotyledons, it can be listed: Poaceae plants (for example, rice, wheat, barley, corn, sugarcane, sorghum, sorghum, lawn grass), Musaceae plants (for example, bananas), Amaryllidaceae plants (for example, green onions, onions, garlic, leeks), Liliaceae plants (for example, lilies, tulips). In addition, among dicotyledonous plants, plants belonging to the family Brassicaceae (e.g., cabbage, radish, Chinese cabbage, rapeseed), the family Asteraceae (e.g., lettuce, burdock, chrysanthemum), the family Fabaceae (e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, licorice), the family Solanaceae (e.g., Plants of the family Cyperaceae (e.g., tomato, eggplant, potato, tobacco, pepper, chili, petunia), plants of the family Rosaceae (e.g., strawberry, apple, pear, peach, loquat, almond, plum, rose, plum blossom, cherry blossom), plants of the family Cucurbitaceae (e.g., cucumber, melon, pumpkin, melon, watermelon), plants of the family Anacardiaceae (e.g., mango, pistachio, cashew), plants of the family Lauraceae (e.g., avocado), plants of the family Rutaceae (e.g., orange, grapefruit, lemon, pomelo), plants of the family Convolvulaceae (e.g., sweet potato), plants of the family Theaceae (e.g., tea tree), and plants of the family Vitaceae (e.g., grape).

[0358] 2-5. Target plant diseases

[0359] The target plant diseases to which the plant disease control composition of the present invention is applied are all plant diseases caused by the target bacteria of the present invention. Preferably, the plant diseases caused by Xanthomonas bacteria are used. For example, bacterial spot (Bacterial spot) visible in peaches, bacterial wilt (Bacterial blight) visible in walnuts, bacterial spot (Bacterial pustu le) visible in soybeans, Angular leaf spot (Angular leaf spot) visible in strawberries, Bacterial blight (Bacrerial blight) visible in tomatoes, peppers, lettuce, etc., Black rot (Black rot) visible in cabbage, Chinese cabbage, cauliflower, etc., Bacterial canker (Bacterial canker) visible in oranges, grapefruits, Angular leaf spot (Angular leaf spot) visible in cotton, Leaf blight (Leaf blight) visible in rice, etc., etc. can be cited.

[0360] 3. Plant disease prevention and control methods

[0361] 3-1. Overview

[0362] The third aspect of the present invention is a method for controlling plant diseases. The method for controlling plant diseases of the present invention is characterized in that the plant disease control composition according to the second aspect is applied to the target plant, thereby controlling the plant disease of the target plant.

[0363] According to the plant disease control method of the present invention, bacterial plant diseases, particularly plant diseases caused by bacteria of the genus Xanthomonas, can be controlled in target plants.

[0364] 3-2. Methods

[0365] The plant disease control method of the present invention includes a contacting step as an essential step.

[0366] The "contacting step" is a step of contacting the plant disease controlling composition according to the second aspect with the target plant. This step is basically in accordance with "2-3. Application method" in the plant disease controlling composition of the second aspect.

[0367] In this embodiment, "contact" means that the plant disease control composition is in contact with the target plant. More specifically, it means that the bacteriolytic agent described in the first embodiment, that is, the bacteriophage, which is an active ingredient of the plant disease control composition, is in contact with the plant body of the target plant, preferably the infected part infected by the target bacteria or the part with a potential infection. The purpose of this step is to allow the bacteriophage as an active ingredient to infect the target bacteria, thereby using it to lyse the target bacteria. As a result, the control effect of plant diseases caused by the target bacteria can be exerted.

[0368] Contact may be direct contact or indirect contact in any manner. In this method, direct contact refers to direct contact between the plant disease control composition and a given part of the target plant. Specifically, for example, it refers to applying, spraying, spreading or immersing a liquid or gel-like plant disease control composition on the plant body of the target plant. In this case, the plant body that is the contact object is mainly leaves, flowers, fruits, stems, branches and / or trunks. On the other hand, indirect contact in this method refers to contact between the plant disease control composition and a given part of the target plant through an intermediary. For example, it refers to applying a granular plant disease control composition to the soil around the roots of the target plant. The bacteriophage as an active ingredient is transported by water in the soil and is quickly absorbed from the roots.

[0369] 3-3. Effect

[0370] The bacteriophage as the active ingredient of the composition obtained by the present invention can efficiently sterilize the target bacteria, thus contributing to the prevention / inhibition of diseases caused by the target bacteria. In addition, the diagnosis of diseases can also be performed by detecting / identifying the target bacteria based on the specific bacteriolytic activity. As the routinely used agents for Xanthomonas bacteria, copper agents and antibiotics can be cited, but these agents can become the main causes of phytotoxicity and destruction of the balance of the flora. For example, it has been confirmed that multiple strains of Pseudomonas fluorescens have plant growth promoting effects (Haas D., Defago G., Nature Rev iews in Microbiology, 2005, 3 (4), 307-19), and copper agents and antibiotics are likely to kill such bacteria that have a symbiotic relationship with plants. On the other hand, bacteriophages are substances of biological origin, so there is no report of obvious phytotoxicity, and they have excellent specificity, so the impact on the balance of the flora is extremely limited.

[0371] 4. Identification of Xanthomonas bacteria

[0372] 4-1. Overview

[0373] A fourth aspect of the present invention is a method for identifying Xanthomonas bacteria. The method of the present invention is characterized in that Xanthomonas bacteria are identified using the host specificity of the bacteriophage constituting the bacteriolytic agent described in the first aspect.

[0374] According to the present invention, it is possible to determine and identify whether or not unidentified plant pathogenic bacteria that cause plant diseases are bacteria belonging to the genus Xanthomonas.

[0375] 4-2. Methods

[0376] The identification method of the present invention includes a culture step, a mixing step, a mixture culture step, and a determination step as essential steps, and further includes a separation step as an optional step.

[0377] (1) Separation process

[0378] The "separation step" is a step of separating the test bacteria from the plant tissues infected by the plant disease. This step is an optional step and can be performed as needed.

[0379] The "test bacteria" refers to plant pathogenic bacteria used in the fourth aspect of the method for identifying Xanthomonas bacteria of the present invention or the fifth aspect of the method for detecting Xanthomonas bacteria described later, and the species of the bacteria is unknown.

[0380] The plant tissue may be any part of a plant that has been infected with a plant disease, and preferably a part where symptoms of the plant disease can be clearly observed. For example, in the case of peach infected with peach bacterial puncture disease, leaves where the disease can be observed can be used.

[0381] In order to isolate the test bacteria from the collected plant tissue, the sample with observed disease can be immersed in a solvent such as water for extraction, and the sample can be fragmented / broken during extraction. Then, the extract is streaked on an agar medium and a single colony is picked.

[0382] (2) Cultivation process

[0383] The "culturing step" is a step of culturing the isolated test bacteria to obtain a culture. The test bacteria can be cultured by a method known in the art.

[0384] The "culture" is obtained by culturing the test bacteria and may be in the form of liquid or solid.

[0385] In this process, the test bacteria are in an unidentified state, so the culture medium used in this process preferably uses a culture medium that can widely culture plant pathogenic bacteria. Use a culture medium that can at least culture the Xanthomonas bacteria that are the identification target bacteria of the present invention. As such a culture medium, for example, as long as it is a culture medium containing one or more components selected from protease decomposition products such as peptone, trypsin, potato glucose, biological source extracts such as yeast extract, amino acids such as glutamic acid or their salts, glucose, sucrose and other sugars, sodium chloride, magnesium chloride, potassium dihydrogen phosphate and other inorganic salts. As specific culture medium and composition, LB culture medium (trypsin, yeast extract, sodium chloride), YPG culture medium (yeast extract, peptone, glucose), PD culture medium (potato glucose), peptone added with Suwa's (Suwa) culture medium (sucrose, glutamic acid, peptone) and the like.

[0386] The isolated test bacteria are inoculated into the above-mentioned culture medium and cultured under appropriate culture conditions. As for the culture conditions, the culture can be obtained by culturing at, for example, 20 to 40°C, 20 to 30°C, 22 to 28°C or 24 to 26°C while stirring. The culture time is not limited, for example, as long as the turbidity at 600nm reaches about 1.0. Through this process, a culture solution of the test bacteria can be obtained. In addition, the culture can be a multi-step culture of more than two steps. For example, a liquid culture medium containing soft agar can be added to the culture solution obtained after culturing in a liquid culture medium, injected into a solid culture medium such as an agar culture medium, and after it is solidified, it is further cultured.

[0387] (3) Mixing process

[0388] The "mixing step" is a step of mixing the culture obtained in the above-mentioned culturing step with the bacteriolytic agent described in the first embodiment to obtain a mixture.

[0389] The "mixture" is a mixture of the culture and the lytic agent, and can be either liquid or solid.

[0390] The mixing method is not particularly limited as long as the culture and the bacteriolytic agent can be mixed. The bacteriolytic agent described in the first aspect may be in a solid state, but may also be administered in a liquid state suspended in water or a liquid culture medium.

[0391] When both the culture medium and the lytic agent are liquid, the volume ratio of the culture medium to the lytic agent can be set to 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 or 9:1. After administration, the culture medium and the lytic agent can be fully mixed by stirring or the like. On the other hand, when a liquid culture medium containing soft agar is layered as described above, the culture medium is solid. In this case, the lytic agent can be dripped onto a solid culture medium such as a gel surface, thereby mixing the two on the solid culture medium to obtain a mixture.

[0392] (4) Mixture Cultivation Step

[0393] The "mixture culturing step" is a step of culturing the above-mentioned mixture under given conditions.

[0394] In addition, when culturing the mixture, a liquid medium containing soft agar may be added to the mixture, and the mixture may be poured onto a solid medium such as an agar medium and allowed to solidify before further culturing.

[0395] The basic steps of this process are based on the above-mentioned culture process. In this process, it is preferred to carry out culture based on the so-called plaque assay method so that it is easy to confirm the presence or absence of lysis of the test bacteria caused by the bacteriophage constituting the lytic agent in the determination process described below, but it is not limited. For example, after a part of the mixed solution is mixed with a soft agar medium of the same composition and before the soft agar medium solidifies, it can be injected on an agar medium of the same composition in the same manner and spread on the entire medium. Then, culture can be carried out under the same conditions as in the above-mentioned culture process.

[0396] (5) Determination process

[0397] The "determination step" is a step of determining that the test bacteria are bacteria of the genus Xanthomonas when the test bacteria undergo lysis after the culturing step.

[0398] The determination of the presence or absence of lysis is not limited. For example, in the case of a plaque assay, it can be determined by the presence or absence of plaque formation. After the above-mentioned mixture culture step, it is developed on an agar medium. In the case where plaques are present on the solidified soft agar medium, it indicates that the test bacteria are lysed due to infection by the bacteriophage constituting the lytic agent of the present invention. Therefore, it can be determined that the test bacteria at this time are Xanthomonas bacteria. On the other hand, in the case where the test bacteria proliferate on the entire agar medium and plaques are completely absent, it can be determined that the test bacteria are not Xanthomonas bacteria.

[0399] In order to make a more accurate judgment, a positive control can be prepared at the same time to confirm that no plaques are produced in the negative control and that plaques can be observed in the positive control. The positive control uses identified Xanthomonas bacteria from the culturing step instead of the negative control and / or test bacteria mixed with the culture medium without a lytic agent in the mixed culturing step.

[0400] 4-3. Effect

[0401] According to the method for identifying Xanthomonas bacteria of the present invention, it is possible to identify whether the cause of plant diseases is Xanthomonas bacteria.

[0402] Furthermore, according to the Xanthomonas bacteria identification method of the present invention, it is possible to detect whether Xanthomonas bacteria exist in the lesion of a plant suffering from a plant disease expected to be caused by Xanthomonas bacteria.

[0403] Example

[0404] The embodiments in the present application correspond to the embodiments in Japanese Patent Application No. 2022-156882 as follows: Embodiment 1 corresponds to Embodiment 1, Embodiment 2 corresponds to Embodiment 2, Embodiment 3 corresponds to Embodiment 3, Embodiment 4 corresponds to Embodiment 4, Embodiment 5 corresponds to Embodiment 5, Embodiment 6 corresponds to Embodiment 6, Embodiment 7 corresponds to Embodiment 7, Embodiment 8 corresponds to Embodiment 8, Embodiment 9 corresponds to Embodiment 9, and Embodiment 10 corresponds to Embodiment 10. In addition, Tables 1 to 13 in the present application are exactly the same as Tables 1 to 13 in Japanese Patent Application No. 2022-156882. In addition, Figures 1 to 11 With Japan's Special Application No. 2022-156882 Figures 1 to 11 In addition, the serial numbers 1 to 44 in the present application are exactly the same as the serial numbers 1 to 44 in Japanese Patent Application No. 2022-156882.

[0405] <Example 1: Isolation of a novel bacteriophage and its bacteriolytic activity (1)>

[0406] (Purpose)

[0407] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases was isolated and its lytic activity against plant pathogenic bacteria was verified.

[0408] (Methods and Results)

[0409] (1) Acquisition and cultivation of plant pathogenic bacteria

[0410] In this example, all the plant pathogenic bacteria used as the target were obtained from the National Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 2 together with the accession number of the National Agricultural Research Organization.

[0411] [Table 2]

[0412] ID Species name Separation source Collection location MAFF No.311019 Xanthomonas oryzae pv.oryzae rice Gifu MAFF No.211965 Xanthomonas oryzoe pv.oryzae rice Okinawa MAFF No.211964 Xanthomonas oryzae pv.oryzae rice Miyazaki MAFF No.211191 Xanthomonas oryzae pv.oryzae rice Miyagi MAFF No.211971 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.311351 Xanthomonas arboricolapv.pruni Peach Fukushima MAFF No.301256 Xanthomonas campestris pv.vesicatoria tomato Tokushima MAFF No.301294 Xanthomonas campestris pv.vesicatoria chili Tokushima MAFF No.301352 Xanthomonas campestris pv.vitians lettuce Wakayama MAFF No.106765 Xanthomonas campestris pv.campestris brocoli Ibaraki MAFF No.301078 Xanthomonas citri subsp. citri Orange Wakayama MAFF No.673005 Xanthomonas cucurbitae pumpkin Hokkaido NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0413] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British microbial strain collection, for which it has been reported to have plant growth-promoting effects and other environmental benefits.

[0414] Xanthomonas oryzae pv.oryzae was cultured using 0.5 g of L-glutamic acid Na, 0.5 g of MgCl 2 6H 2 O1.0g, KH 2 PO 4 0.1g, 5g sucrose and 5g peptone were dissolved in H 2 1 L of autoclaved liquid medium (SW+P Broth) was used for culturing other Xanthomonas bacteria and Pseudomonas fluorescens. 1 g of peptone, 1 g of yeast extract, and 2 g of glucose were dissolved in H 2 O 1L and autoclaved liquid culture medium (YPGBroth). In addition, as an agar culture medium, an agar culture medium prepared by adding 15 g of agar per 1L to the above Broth (SW+P Broth or YPG Broth) and autoclaving was used (indicated as "SW+P Agar" when SW+P Broth was used, and "YPG Agar" when YPG Broth was used). In addition, a soft agar culture medium (Top Agar) layered on the agar culture medium was prepared by adding 5 g of agarose per 1L to the above Broth and autoclaving it at about 50°C, and was used as needed.

[0415] After suspending each of the above strains delivered in a dry powder state in 0.1 mL of Broth, a line culture was performed at 25°C using Agar (SW+PAgar or YPG Agar) to isolate a single colony. The isolated colony was inoculated in Broth and cultured with shaking at 25°C to serve as a pre-culture solution. In this culture, the pre-culture solution was inoculated in Broth and cultured at 25°C for 10 to 30 hours until the turbidity (Optical Density 600nm) reached about 1.0. The culture solution after culture was used directly as a bacterial solution.

[0416] (2) Isolation and purification of the first phage

[0417] A new bacteriophage was isolated from natural sewage or soil obtained in Japan. The method for isolating bacteriophages is based on the conventional plaque assay method. First, sewage from ponds, lakes, etc., or sewage obtained by suspending soil in water is filtered with a 0.45μm filter to prepare a liquid containing bacteriophages. Then, the bacterial liquid and the liquid containing bacteriophages are mixed in equal amounts and left at room temperature for about 10 minutes. Next, 0.2mL of the bacteria / bacteriophage mixture is added to 3mL of TopAgar, quickly mixed with a vortex mixer, and then injected into the Agar. After Top Agar solidifies, it is cultured at 25°C for about 12 hours. Lytic plaques are formed on the bacterial lawn (Lawn) formed by the culture. Then, the gel of the plaque part is sucked up using a pipette tip with a front end cut off, and bacteriophages with lytic activity against Xanthomonas bacteria are separated. Then, the bacteriophage-containing liquid containing the isolated bacteriophages at a high concentration is used instead of sewage, and this step is repeated to purify the bacteriophages.

[0418] A total of three new phages were isolated from natural sewage / soil by detecting plaques formed on soft agar medium in which any of the above strains were amplified. The three new phages isolated in Example 1 are referred to as "first phages".

[0419] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the above bacterial solution under the above conditions, and the phages were separated again. By repeating this step several times, the phages were further purified. The composition of SM Buffer is shown in Table 3.

[0420] [Table 3]

[0421] SM Buffer final Add to 1L NaCl 0.1M 5.8g <![CDATA[Mg 2 SO 4 ·7H 2 O]]> 10mM 1g 1M Tris-HCl pH 8.0 50mM 50mL gelatin 0.1％ 0.1g

[0422] (3) Amplification and purification of the first phage

[0423] In order to amplify and purify the first phage after separation and purification, an amplification method using a plaque assay, i.e., a plate lysate (PL) method, was implemented. A bacteria / phage mixed solution was prepared in such a way that a large number of plaques were formed on Agar, and after mixing with Top Agar, it was developed on YPG Agar and cultured. Then, 3 mL of SM Buffer was added to the Top Agar where plaques were formed, and the mixture was shaken at 25°C for about 30 minutes, and the supernatant was passed through a 0.2 μm filter to recover the recovery solution containing the phage.

[0424] In the purification of the first phage, PEG 60001g (final concentration 10%) and NaCl 0.4g (final concentration 4%) were added and dissolved in 10mL of the above recovery solution, and the solution was rotated overnight at 4°C using a rotator. Then, centrifugation was performed at ×15,000e / 4°C / 60 minutes, and the supernatant was removed. The recovered particles were resuspended in 0.5mL of SM Buffer. Next, 0.5mL of chloroform was added, stirred vigorously, and placed on ice for 6 hours. After centrifugation at ×8,000g / 4°C / 10 minutes, the upper layer was carefully recovered to obtain a phage purified solution. The concentration of the phage purified solution is usually expressed as a titer [PFU / mL] based on the number of plaques (Plaque Forming Unit, PFU) in the plaque assay as an indicator of bacteriolytic activity. The titer of the prepared first phage purified solution was determined by a plaque assay using an appropriately diluted solution, and was confirmed to be 10 8 PFU / mL or above.

[0425] (4) Evaluation of the host range of the first phage

[0426] The host range of the first phage was evaluated by the spot test method. Only 0.1 mL of the bacterial solution was added to 3 mL of Top Agar and mixed, then injected into Agar, spread over the entire plate, and allowed to solidify. In addition to the bacterial solutions of each Xanthomonas bacterium shown in Table 1 prepared in (1) above, a bacterial solution of Pseudomonas fluorescens was also prepared for control. Then, about 5 μL of the purified phage solution was added dropwise, and the mixture was incubated at 25°C for about 12 hours. On the plate where a bacterial lawn of bacteria was formed, if the area where the solution was added becomes transparent (clear) in a circular shape (about 1 cm in diameter), it is determined that the added phage has bacteriolytic activity against the strain.

[0427] An example of the results is shown in Figure 1. When the first phage shows lytic activity, in the bacterial moss formed on the plate, only the part where the phage purification solution was dropped becomes transparent. The three first phages obtained by the present invention show lytic activity against all strains of the five bacterial species Xanthomonas arboricola, Xanthom onas campestris, Xanthomonas citri, Xanthomonas oryzae and Xanthomonas cucurbitae shown in Table 2. On the other hand, it was also confirmed that no lytic activity was shown against Pseudomonas fluorescens. Although there are reports of phages showing activity against two or more species of Xanthomonas, such a combination as mentioned above has not been reported (Nakayinga R. et al., BMC Microbiology, 2021, 21: 291).

[0428] An example of a plate for plaque assay for the first phage having the base sequence shown in SEQ ID NO: 8 using Xanthomonas oryzae pv. Oryzae with ID MAFF No. 311019 as a bacterial species is shown in FIG. Fig.12 As a result of the lysis, a large number of plaques were formed, indicating that the first phage showed lytic activity against the bacteria isolated from rice. These results indicate that the first phage has the potential to be applied to various plant diseases. For example, it is suggested that it is useful for the prevention and treatment of peach bacterial puncture disease, rice bacterial blight, citrus canker, and cauliflower black rot, which are caused by Xanthomonas bacteria, and it is expected that the value for industrial use is also high.

[0429] (5) Genome analysis of the first phage

[0430] The genomic DNA sequence of the first phage was determined and analyzed.

[0431] (i) Preparation and sequence determination of genomic DNA of the first phage

[0432] Using TURBO DNA-free TMThe genome of the first phage was extracted using the kit (Thermo Fisher Scientific). The genomic DNA from the host bacteria that became an inclusion was removed by processing according to the manual attached to the kit. Then, using NucleoSpin (registered trademark) Virus (Mac hery-Nagel), the phage capsid molecules were decomposed by proteinase K treatment according to the attached manual. After genomic DNA purification using a silica spin column, a genomic DNA solution of the first phage was prepared. Then, the concentration of genomic DNA was determined using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific), and 50 μL of a genomic DNA solution with a final concentration of 0.2 ng / μL was prepared. Next, using Nextera XT DNA Library Prep (Illumi na), the genome of the first phage was fragmented and the linker sequence was added based on PCR by processing according to the attached manual. Next, using Agilent High Sensitivity DNA Kit (Agilent Technologies), electrophoresis based on Bioanalyzer (Agilent Technologies) was performed to measure the average bp size of the sample and the concentration of the DNA fragment was obtained. Finally, using Miseq Reagent kit (Illumina), the sample for measurement was prepared by processing according to the attached manual, and the measurement was performed on the next generation sequencer Miseq (Illumina). Using CLC genomics workbench (Qiagen), the obtained data was pre-processed (trimmed, etc.), and then de novo assembly was performed to obtain a conti g sequence equivalent to the genomic sequence of the bacteriophage.

[0433] (ii) Bioinformatics analysis based on genome sequence information

[0434] The genome base sequences of the first phage (sequence numbers 8 to 10) were subjected to BLAST analysis using GENETYX-NGS installed in GENETYX, and the results showed that the base sequences of sequence numbers 8 to 10 had high sequence identity with each other. For example, the sequence identity (average nucleotide identity (Average Nucleotide Identity: ANI)) of the base sequence of sequence number 8 with respect to the base sequence of sequence number 9 was 98% or more within a range of 96% or more of the entire length, and the sequence identity of the base sequence of sequence number 8 with respect to the base sequence of sequence number 10 was 98% or more within a range of 95% or more of the entire length.

[0435] Furthermore, the tail fiber gene was searched from the genome sequence information of the three phages mentioned above. The tail fiber gene was searched using the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ), and the base sequences shown in sequence numbers 5 to 7, which were presumed to be the tail fiber genes, were determined from the genome sequences of each phage. The base sequences of sequence numbers 5 to 7 were analyzed using GENETYX, and the results showed that the base sequences of sequence numbers 5 to 7 had high sequence identity with each other. For example, the sequence identity of the base sequence of sequence number 5 with respect to the base sequences of sequence numbers 6 and 7 was 96% or more, and the sequence identity of the base sequences of sequence numbers 6 and 7 was 98% or more. In addition, the amino acid sequences (sequence numbers 2 to 4) encoded by the base sequences of sequence numbers 5 to 7 were analyzed using GENETYX, and the results showed that the amino acid sequences of sequence numbers 2 to 4 also had high sequence identity with each other, and the sequence identity between the amino acid sequences of sequence numbers 2 to 4 was 98% or more.

[0436] Based on the genome base sequences of the above-mentioned bacteriophages (sequence numbers 8 to 10), a search for similar DNA sequences and confirmation of sequence identity were performed using the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The search results showed that the genome sequence of the bacteriophage Mija described in U.S. Patent Application Publication No. 2016 / 0309723 (sequence number 29 of the above-mentioned publication) had the highest kinship score, for example, with respect to the genome base sequence of sequence number 8, it had a sequence identity of 86.5% over 93% of the total length.

[0437] Xylella fastidiosa et al. confirmed the lytic activity of phage Mija, and the host range of phage Mija is different from that of the phage of the present invention. Generally, regarding the host specificity and host range characteristics of phages, it is believed that the tail fiber protein plays a great role (Nobrega FL et al., Nat. Rev. Microbiol., 2018, 16: 760-773), therefore, the present inventors believe that the above-mentioned difference in host range is due to the difference in tail fiber genes.

[0438] Therefore, using the base sequence of a specific tail fiber gene (sequence numbers 5 to 7) as a query, similar DNA sequences were searched in the genome sequence of bacteriophage Mija. As a result, in the genome sequence of bacteriophage Mija, a region with 83% sequence identity to the base sequence shown in sequence number 5 was found only within 16% of the total length, and no DNA sequence identical to the base sequence shown in sequence numbers 5 to 7 was found in the entire length. According to this result, the difference in the host range of bacteriophage Mija and the three bacteriophages obtained in the examples of this application is due to the difference in the tail fiber gene.

[0439] In order to investigate whether the tail fiber gene possessed by the bacteriophage of the present invention is new, the amino acid sequences of sequence numbers 2 to 4 were used as queries, and similar amino acid sequences were searched using the BLAST server provided by NCBI. As a result, the tail fiber protein of Stenotrophomonas phage vB_SmaS-DLP_6 (GenBank accession number: AMQ65898.1) had the highest kinship score. However, the sequence identity of its amino acid sequence to any of the amino acid sequences in sequence numbers 2 to 4 is low. For example, relative to the sequence of sequence number 2, it has a sequence identity of 58.47% only within 35% of the total length. It should be noted that Stenotrophomonas phage vB_SmaS-DLP_6 targets the genus Stenotrophomonas, which is a different genus from the bacteriophage of the present invention.

[0440] The above results indicate that the phage of the present invention has a novel host range of Xanthomonas that has not been reported before, and its characteristics are particularly based on the novel tail fiber gene.

[0441] <Example 2: Isolation of a new bacteriophage and its bacteriolytic activity (2)>

[0442] (Purpose)

[0443] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0444] (Methods and Results)

[0445] (1) Acquisition and cultivation of plant pathogenic bacteria

[0446] In this example, the plant pathogenic bacteria used as the target bacteria were Xanthomonas bacteria, and all strains were obtained from the Agricultural Research Organization (NARO). The bacteria used in Example 2 are listed in Table 4 together with their respective NARO accession numbers (MAFF No.).

[0447] [Table 4]

[0448] ID Species name Separation source Collection location MAFF No.311019 Xanthomonas oryzae pv.oryzae rice Gifu MAFF No.211965 Xanthomonas oryzae pv.oryzae rice Okinawa MAFF No.211964 Xanthomonas oryzae pv.oryzae rice Miyazaki MAFF No.211191 Xanthomonas oryzae pv.oryzae rice Miyagi MAFF No.211971 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.311351 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.212146 Xanthomonas arboricola pv.juglandis Walnut Nagano MAFF No.301256 Xanthomonas campestris pv.vesicatoria tomato Tokushima MAFF No.301294 Xanthomonas campestris pv.vesicatoria chili Tokushima MAFF No.301352 Xanthomonas campestris pv.vitians lettuce Wakayama MAFF No.301078 Xanthomonas citri subsp. citri Orange Wakayama MAFF No.673005 Xanthomonas cucurbitae pumpkin Hokkaido NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0449] In addition, as a control, a strain of Pseudomonas fluorescens (NCIM B-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0450] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0451] (2) Isolation and purification of the second phage

[0452] A new bacteriophage was isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, a new bacteriophage was isolated from natural sewage / soil. The new bacteriophage isolated in Example 2 is referred to as "second bacteriophage".

[0453] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0454] (3) Amplification and purification of the second phage

[0455] In order to amplify and purify the separated and purified second phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the first phage" of Example 1. The titer of the prepared second phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0456] (4) Evaluation of the host range of the second phage

[0457] The host range of the second phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0458] An example of the results is shown in Figure 2 . When the second phage exhibits lytic activity, in the bacterial moss formed on the plate, only the portion to which the phage purification solution was dropped becomes transparent. The second phage obtained by the present invention exhibits lytic activity against all Xanthomonas bacteria shown in Table 4. On the other hand, it does not exhibit lytic activity against Pseudomonas fluorescens. As in Example 1, a phage that exhibits lytic activity against all of the multiple bacterial species shown in Table 4 is not known to date.

[0459] An example of a plate for plaque assay using Xanthomonas or yzaepv. Oryzae with ID MAFF No. 311019 as a bacterial species for the second phage is shown in FIG. Fig.13 As a result of the lysis, a large number of plaques were formed, indicating that the second phage also exhibited lytic activity against the bacteria isolated from rice. This result suggests that the isolated second phage exhibited lytic activity against a wide range of Xanthomonas bacteria.

[0460] In addition, the results suggest that the second phage is useful for controlling peach bacterial borer disease, rice bacterial blight, citrus canker, cauliflower black rot, and the like, which are caused by Xanthomonas bacteria.

[0461] (5) Genome analysis of the second phage

[0462] The genomic DNA sequence of the second phage was determined and analyzed.

[0463] (i) Preparation and sequence determination of genomic DNA of the second phage

[0464] Using TURBO DNA-free TM The genome of the second phage was extracted using a kit (Thermo Fisher Scientific). The basic operation was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0465] (ii) Bioinformatics analysis based on genome sequence information

[0466] Based on the genomic base sequence of the phage obtained in (i) above (sequence number 13), a search for similar DNA sequences and confirmation of sequence identity were performed using the BLAST server provided by NCBI (same as above). As a result of the search, the genomic sequence of the phage targeting the Xanthomonas bacterium called Xp12 (accession number: MT664984.1) had the highest affinity score, with a sequence identity of 98.42%. However, the only bacterial species known to exhibit lytic activity against phage Xp12 is Xanthomonas oryzae (Nakayinga R. et al., BMC Microbiology, 2021, 21: 291).

[0467] In order to clarify the reason why Xp12 and the second phage isolated in this example have different host specificities, the sequences of the fiber proteins of the two phages were compared.

[0468] First, the fiber gene was identified from the genome sequence of the second phage of 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 base sequence shown in sequence number 12 was determined as the fiber gene.

[0469] Furthermore, the amino acid sequence (SEQ ID NO. 11) encoded by the gene containing the base sequence shown in SEQ ID NO. 12 was compared with the amino acid sequence of a protein related to Xp12 (accession number: QNN97189.1), and the results showed that the types of amino acids at positions 278 and 350 were different. Specifically, the valine at position 278 in Xp12 was changed to alanine, and the serine at position 350 was changed to tryptophan. Since these are not conservative substitutions, and serine in particular contributes to protein-protein interactions in many cases, there is a high possibility that these substitutions will cause differences in function.

[0470] Based on the above results, it can be inferred that the second phage has a new host range of Xanthomonas that has not been reported before, and its characteristics are based on the tail fiber gene.

[0471] <Example 3: Isolation of a new bacteriophage and its bacteriolytic activity (3)>

[0472] (Purpose)

[0473] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0474] (Methods and Results)

[0475] (1) Acquisition and cultivation of plant pathogenic bacteria

[0476] In this example, the plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 5 together with their respective NARO accession numbers (MAFF No.).

[0477] [Table 5]

[0478] ID Species name Separation source Collection location MAFF No.311282 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.301426 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311351 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311586 Xanthomonas arboricola pv.pruni Peach Nagano MAFF No.311618 Xanthomonas arboricola pv.pruni Peach Wakayama NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0479] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0480] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0481] (2) Isolation and purification of phage 3

[0482] A new bacteriophage was isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, a new bacteriophage was isolated from natural sewage / soil. The new bacteriophage isolated in Example 3 is referred to as "the third bacteriophage".

[0483] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0484] (3) Amplification and purification of the third phage

[0485] In order to amplify and purify the isolated and purified third phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the first phage" of Example 1. The titer of the prepared third phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0486] (4) Evaluation of the host range of phage 3

[0487] The host range of the third phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0488] An example of the results is shown in Figure 3 . When the third phage shows lytic activity, in the bacterial moss formed on the plate, only the part where the phage purification solution was dropped becomes transparent. The third phage obtained by the present invention shows lytic activity against all Xanthomonas bacteria shown in Table 5. On the other hand, it does not show lytic activity against Pseudomonas fluorescens. It is generally known that the host range of bacteriophages 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, as shown in the collection location of Table 5, the phage isolated in this example shows lytic activity against any Xanthomonas bacteria isolated in different regions. This suggests that the isolated third phage shows lytic activity against a wide range of Xanthomonas bacteria.

[0489] In addition, these results suggest that the third phage is useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0490] (5) Genome analysis of phage 3

[0491] The genomic DNA sequence of the third phage was determined and analyzed.

[0492] (i) Preparation and sequence determination of genomic DNA of phage 3

[0493] Using TURBO DNA-free TM The genome of the third phage was extracted using a kit (Thermo Fisher Scientific). The basic operation was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0494] (ii) Bioinformatics analysis based on genome sequence information

[0495] Based on the genome base sequence (sequence number 14) of the third phage obtained in (i) above, the BLAST server provided by NCBI (same as above) was used to search for similar DNA sequences and confirm the sequence identity. As a result of the search, the kinship scores of various Pseudomonas phage complete genomes were the highest. (Accession numbers: KX129925.1, MN504636.1, KX898399.1, NC_041953.1, MW835180.1). However, only about 75% sequence identity was observed in any sequence within an extremely local range of 6% to 7% of the total genome length. Moreover, the bacteria that serve as hosts for these phages are different from those of the genus Xanthomonas. The results suggest that the third phage is a phage with a new genomic sequence whose kinship genomic sequence is completely unknown.

[0496] <Example 4: Isolation of a new bacteriophage and its bacteriolytic activity (4)>

[0497] (Purpose)

[0498] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0499] (Materials and methods)

[0500] (1) Acquisition and cultivation of plant pathogenic bacteria

[0501] In this example, the plant pathogenic bacteria used as the target bacteria were Xanthomonas bacteria, and all strains were obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 6 together with their respective NARO accession numbers (MAFF No.).

[0502] [Table 6]

[0503] ID Species name Separation source Collection location MAFF No.311282 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311351 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311417 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311579 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311584 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311586 Xanthomonas arboricola pv.pruni Peach Nagano MAFF No.311618 Xanthomonas arboricola pv.pruni Peach Wakayama MAFF No.311641 Xanthomonas arboricola pv.pruni Peach Fukushima NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0504] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0505] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0506] (2) Isolation and purification of phage 4

[0507] A new bacteriophage was isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, a new bacteriophage was isolated from natural sewage / soil. The new bacteriophage isolated in Example 4 is referred to as "the fourth bacteriophage."

[0508] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0509] (3) Amplification and purification of phage 4

[0510] In order to amplify and purify the fourth phage after separation and purification, an amplification method using a plaque assay, i.e., a plate lysate (PL) method, was performed. The specific method was based on the method described in "(3) Amplification and purification of the first phage" of Example 1. The titer of the prepared fourth phage purified solution was determined by a plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0511] (4) Evaluation of the host range of phage 4

[0512] The host range of the fourth phage was evaluated by the spot test method. The basic operation was based on 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 . When the 4th phage showed lytic activity, in the bacterial moss formed on the plate, only the part to which the phage purification solution was dropped became transparent. The 4th phage obtained by the present invention showed lytic activity against all strains of Xanthomonas bacteria shown in Table 6. As in Example 3, lytic activity was shown against any Xanthomonas bacteria isolated from different regions. This suggests that the isolated phage showed lytic activity against a wide range of Xanthomonas bacteria.

[0513] In addition, these results suggest that phage 4 is useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0514] (5) Genome analysis of phage 4

[0515] The genomic DNA sequence of the fourth phage was determined and analyzed.

[0516] (i) Preparation and sequence determination of genomic DNA of phage 4

[0517] Using TURBO DNA-free TM The phage genome was extracted using a kit (Thermo Fisher Scientific). The basic procedure was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0518] (ii) Bioinformatics analysis based on genome sequence information

[0519] Based on the genomic base sequence of the phage obtained in (i) above (sequence number 17), the BLAST server provided by NCBI (same as above) was used to implement the search for similar DNA sequences and confirmation of sequence identity. The result of the search was that some phages named FoX isolated in Belgium recorded by Nakayinga et al. (Nakayinga R. et al., BMC Microbiology, 2021, 21: 291) (inclusion numbers: NC_055835.1, NC_055836.1, NC_055837.1, NC_055838.1) had the highest affinity score. However, with respect to any sequence, there is only about 77% sequence identity within a range of about 10% of the total length. This result suggests that the phage isolated in Example 1 is basically a phage with a new genomic sequence for which there is no report of a related sequence.

[0520] Generally, regarding the host specificity and host range characteristics of bacteriophages, it is believed that the tail fiber protein plays a major role (Nobrega F. Let al., Nat. Rev. Microbiol., 2018, 16: 760-773). Therefore, the tail fiber gene was retrieved from the genome sequence information of the above-mentioned 4th phage. The base sequence shown in sequence number 16, which is presumed to be the tail fiber gene, was determined from the genome sequence of the 4th phage using the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ). The identity of this gene sequence with the base sequence of the tail fiber gene of the known phage Fox is 58%. The host species of the reported phage Fox is Xanthomonas campestris (Nakayinga R. et al., BM C Microbiology, 2021, 21: 291). Therefore, it is different from the host range of the 4th phage.

[0521] In addition, this result suggests that it is difficult to predict the host range of phages having fiber genes with different base sequences based on the correlation between known fiber gene information and host species information of phages having the gene.

[0522] Based on the above results, it is estimated that the fourth phage has a new host range of Xanthomonas that has not been reported before, and its characteristics are particularly based on the new tail fiber gene.

[0523] <Example 5: Isolation of a new bacteriophage and its bacteriolytic activity (5)>

[0524] (Purpose)

[0525] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0526] (Methods and Results)

[0527] (1) Acquisition and cultivation of plant pathogenic bacteria

[0528] In this example, the plant pathogenic bacteria used as the target bacteria were Xanthomonas bacteria, and all strains were obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 7 together with their respective NARO accession numbers (MAFF No.).

[0529] [Table 7]

[0530] ID. Species name Separation source Collection location MAFF No.211971 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.31 1351 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311622 Xanthomonas arboricola pv.pruni Peach Yamanashi NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0531] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0532] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0533] (2) Isolation and purification of phage 5

[0534] New bacteriophages were isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, two new bacteriophages were isolated from natural sewage / soil. The new bacteriophage isolated in Example 5 is referred to as "the fifth bacteriophage."

[0535] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0536] (3) Amplification and purification of phage 5

[0537] In order to amplify and purify the isolated and purified 5th phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the 1st phage" of Example 1. The titer of the prepared 5th phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0538] (4) Evaluation of the host range of phage 5

[0539] The host range of the fifth phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0540] An example of the results is shown in Figure 5 . When the 5th phage showed lytic activity, in the bacterial moss formed on the plate, only the part to which the phage purification solution was dropped became transparent. The 5th phage obtained by the present invention showed lytic activity against all Xanthomonas bacteria shown in Table 7. On the other hand, it did not show lytic activity against Pseudomonas fluorescens. As in Example 3, lytic activity was shown against any Xanthomonas bacteria isolated from different regions. This suggests that the isolated 5th phage showed lytic activity against a wide range of Xanthomonas bacteria.

[0541] In addition, these results suggest that phage 5 is useful for controlling bacterial blowhole of peach caused by bacteria of the genus Xanthomonas.

[0542] (5) Genome analysis of phage 5

[0543] The genomic DNA sequence of phage 5 was determined and analyzed.

[0544] (i) Preparation and sequence determination of phage 5 genomic DNA

[0545] Using TURBO DNA-free TMThe genome of the fifth phage was extracted using a kit (Thermo Fisher Scientific). The basic operation was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0546] (ii) Bioinformatics analysis based on genome sequence information

[0547] Based on the genomic base sequence of the 5th phage obtained in (i) above (sequence numbers 18 and 19), the BLAST server provided by NCBI (same as above) was used to implement the search for similar DNA sequences and confirmation of sequence identity. As a result of the search, a large number of related sequences were hit. The sequence with the highest sequence identity within a range of more than 90% of the total length is Pseudomonas phage vB_PaeS (accession number: LC552830.1) in the case of sequence number 18, and P1940 (accession number: MX298177.1) described in the U.S. Patent Application Publication No. 2017-0319637 in the case of sequence number 19. Each sequence is shown in Table 8 below, and the same degree of sequence identity is shown in both sequence numbers 18 and 19.

[0548] [Table 8]

[0549]

[0550] In the table, sequence identity is a numerical value relative to the range of automatic matching of the analysis server to the full length of the genome of sequence number 18 or 19. The numerical value of this range is expressed as Query Cover. For example, the sequence identity of vB_PaeS of 98.28% is a numerical value calculated by limiting the region of 90% of the full length of sequence number 18. Therefore, although it is difficult to calculate the sequence identity relative to the full length, it can be inferred that it is at least less than 98.28%, which is equivalent to a numerical value less than 90%. In the case of P1940, it can be inferred that it is equivalent to a numerical value less than 95%.

[0551] As a result of this analysis, it was clarified 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 sequence identity of the 5th phage of this example is extremely high with the genome sequence of the known phage, it is difficult to determine that it is its target bacteria. At the same time, it can be considered that for the 5th phage, the phage that shows higher sequence identity in a wider range than vB_PaeS and P1940 has a high possibility of having the same host range as the 5th phage of this example, that is, bacteria of the genus Xanthomonas.

[0552] Based on the above results, it can be inferred that the fifth phage has a new host range of the genus Xanthomonas that has not been reported before.

[0553] <Example 6: Isolation of a new bacteriophage and its bacteriolytic activity (6)>

[0554] (Purpose)

[0555] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0556] (Methods and Results)

[0557] (1) Acquisition and cultivation of plant pathogenic bacteria

[0558] In the present invention, the plant pathogenic bacteria used as target bacteria are Xanthomonas bacteria, and all strains are obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 9 together with their respective NARO accession numbers (MAFF No.).

[0559] [Table 9]

[0560] ID Species name Separation source Collection location MAFF No.211971 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.311351 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311622 Xanthomonas arboricola pv.pruni Peach Yamanashi NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0561] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0562] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0563] (2) Isolation and purification of phage 6

[0564] A new bacteriophage was isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, a new bacteriophage was isolated from natural sewage / soil. The new bacteriophage isolated in Example 6 is referred to as "the sixth bacteriophage".

[0565] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0566] (3) Amplification and purification of phage 6

[0567] In order to amplify and purify the isolated and purified 6th phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the 1st phage" of Example 1. The titer of the prepared 6th phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0568] (4) Evaluation of the host range of phage 6

[0569] The host range of the sixth phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0570] An example of the results is shown in Figure 6 . When the 6th phage showed lytic activity, in the bacterial moss formed on the plate, only the part to which the phage purification solution was dropped became transparent. The 6th phage obtained by the present invention showed lytic activity against all Xanthomonas bacteria shown in Table 9. On the other hand, it did not show lytic activity against Pseudomonas fluorescens. As in Example 3, lytic activity was shown against any Xanthomonas bacteria isolated from different regions. This suggests that the isolated 6th phage showed lytic activity against a wide range of Xanthomonas bacteria.

[0571] In addition, these results suggest that phage 6 is useful for controlling bacterial blowhole disease of peach caused by bacteria of the genus Xanthomonas.

[0572] (5) Genome analysis of phage 6

[0573] The genomic DNA sequence of the sixth phage was determined and analyzed.

[0574] (i) Preparation and sequence determination of genomic DNA of phage 6

[0575] Using TURBO DNA-free TM The genome of the sixth phage was extracted using a kit (Thermo Fisher Scientific). The basic procedure was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage."

[0576] (ii) Bioinformatics analysis based on genome sequence information

[0577] Based on the genome base sequence (sequence number 23) of the 6th phage obtained in (i) above, the BLAST server provided by NCBI (same as above) was used to implement the search for similar DNA sequences and the confirmation of sequence identity. The result of the search was that 4 phages (accession numbers: NC_017864.1, NC_007806.1, NC_029019.1, NC_024381.1) showed 96.5-98.5% sequence identity within the range of about 90% of the full length of the genome. It can be inferred that the sequence identity relative to the full length is equivalent to a numerical value of about 85%. However, despite the high sequence identity, the target bacteria of these known phages are not Xanthomonas, but Pseudomonas or Stenotrophomonas. Therefore, it is suggested that the 6th phage is a brand new phage as a phage that targets Xanthomonas bacteria.

[0578] Therefore, in order to identify genes related to target bacteria, the genome sequences of the sixth phage were compared with the above four known phages. Furthermore, ORFs were identified in the regions with low sequence identity to the known phages found by the above comparison. The identification of ORFs utilized the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ).

[0579] Through the above comparison, it was found that the 8062nd to 9473rd and 31995th to 34230th ranges in the genomic base sequence shown in sequence number 23 are regions with low sequence identity to known bacteriophages. A total of 5 ORFs were detected in this region. The specific ORFs are DNA regions shown in the base sequences of the 8082nd to 8573rd, 8645th to 8842nd, 9223rd to 9477th, 31881st to 32789th and 32792nd to 33694th positions in the genomic base sequence of sequence number 23, respectively.

[0580] In order to clarify which gene is important for determining the host range, each ORF was investigated in detail. As a result, for known phages targeting Xanthomonas bacteria, base sequence hits with a sequence identity of 30% or more were the most, so it was estimated that in the 6th phage isolated in Example 6, the base sequence of positions 9223 to 9477 (sequence number 22) in the genomic base sequence shown in sequence number 23 showed high sequence identity and was an important gene region.

[0581] In order to clarify whether the gene is a known gene, based on the amino acid sequence of the translation product (SEQ ID NO: 21), a broad search for similar amino acid sequences was performed using the BLAST server provided by NCBI, not limited to phages targeting Xanthomonas bacteria. As a result, even with the highest affinity score, the sequence identity was only about 50%, and no sequence showing high sequence identity was detected.

[0582] <Example 7: Isolation of a new bacteriophage and its bacteriolytic activity (7)>

[0583] (Purpose)

[0584] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0585] (Methods and Results)

[0586] (1) Acquisition and cultivation of plant pathogenic bacteria

[0587] In the present invention, the plant pathogenic bacteria used as target bacteria are Xanthomonas bacteria, and all strains are obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 10 together with their respective NARO accession numbers (MAFF No.).

[0588] [Table 10]

[0589] ID Species name Separation source Collection location MAFF No.211971 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.311351 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311622 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.106765 Xanthomonas campestris pv.campestris brocoli Ibaraki MAFF No.106642 Xanthomonas campestris pv.campestris Fescue Ibaraki NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0590] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0591] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0592] (2) Isolation and purification of phage 7

[0593] New bacteriophages were isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, two new bacteriophages were isolated from natural sewage / soil. The new bacteriophage isolated in Example 7 is referred to as "the seventh bacteriophage".

[0594] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0595] (3) Amplification and purification of phage 7

[0596] In order to amplify and purify the isolated and purified phage No. 7, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of phage No. 1" of Example 1. The titer of the prepared phage No. 7 purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0597] (4) Evaluation of the host range of phage 7

[0598] The host range of the seventh phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0599] An example of the results is shown in Figure 7 . When the 7th phage showed lytic activity, in the bacterial moss formed on the plate, only the part to which the phage purification solution was dropped became transparent. The 7th phage obtained by the present invention showed lytic activity against all Xanthomonas bacteria shown in Table 10. On the other hand, it did not show lytic activity against Pseudomonas fluorescens. As in Example 1, it showed lytic activity against all the various bacterial species and also against strains isolated from different regions. This suggests that the isolated 7th phage showed lytic activity against a wide range of Xanthomonas bacteria.

[0600] In addition, these results suggest that phage 7 is useful for controlling bacterial blowhole disease of peach and black rot of cauliflower caused by bacteria of the genus Xanthomonas.

[0601] (5) Genome analysis of phage 7

[0602] The genomic DNA sequence of phage 7 was determined and analyzed.

[0603] (i) Preparation and sequence determination of genomic DNA of phage 7

[0604] Using TURBO DNA-free TMThe genome of the seventh phage was extracted using a kit (Thermo Fisher Scientific). The basic procedure was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage."

[0605] (ii) Bioinformatics analysis based on genome sequence information

[0606] Based on the genomic base sequence of the 7th phage obtained in (i) above (sequence numbers 28 and 29), the BLAST server provided by NCBI (same as above) was used to implement the search for similar DNA sequences and confirmation of sequence identity. As a result of the search, the genomic sequence of the phage recorded in Japanese Patent Gazette No. 2021-102635 (sequence numbers 19 and 20 in the document (sequence numbers 30 and 31 in the specification of this application, respectively)) had the highest kinship score. Any sequence has a sequence identity of approximately 92% within a range of approximately 90% of the total length. Based on this result, it can be inferred that the sequence identity relative to the full length is equivalent to a value of about 85%. In addition, the kinship score of the genomic base sequence of ΦXc10 (Nakayinga R. et al., BMC M icrobiology, 2021, 21: 291) is the second highest. The genomic base sequence of the above-mentioned ΦXc10 (accession number: NC_047840.1) has a sequence identity of about 92% within 87% of the total length. According to the result, it can be inferred that the sequence identity relative to the full length is equivalent to a value of about 80%. In addition, the kinship scores of the genomic base sequences of f30-Xaj and f20-Xaj (NakayingaR.et al., 2021, ibid.) are also high. The genomic base sequences of the above-mentioned f30-Xaj and f20-Xaj (accession numbers: NC_030937.1 and NC_030928.1) have a sequence identity of about 91% within 74% of the total length. According to the result, it can be inferred that the sequence identity relative to the full length is equivalent to a value of about 70%. However, any phage is not a phage that uses Xanthomonas arboricola pv.Pruni as a target bacterium.

[0607] In order to explore the reasons for the differences in target bacteria, a search for new genes was conducted in the genomic base sequence information of the 7th phage (sequence number 28). The inference of the gene region utilized the RAST server (http: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ). As a result of the exploration, a gene group encoding a new tail tubular protein was identified as a gene related to the host range of the 7th phage. For their gene regions, in the genomic base sequences of the two 7th phages obtained in (i) above, the base sequences exist in the same region.

[0608] Sequence number 26 represents the base sequence of the tail tube protein A gene contained in the genome base sequence (sequence number 28) of the seventh phage discovered in this example, and sequence number 24 represents the amino acid sequence of the tail tube protein A. On the other hand, sequence number 27 (to be precise, sequence number 58) represents the base sequence of the tail tube protein B gene, and sequence number 25 (to be precise, sequence number 57) represents the amino acid sequence of the tail tube protein B.

[0609] Since it has been reported that tail tube protein A and tail tube protein B are related to the specific adsorption of phage to bacteria (Maozhi Hu, et ai., 2020, 9: 1, 855-867), it is highly likely that the seventh phage is also related to the host range of the phage. However, based on the amino acid sequence of tail tube protein A (sequence number 24) and the amino acid sequence of tail tube protein B (sequence number 25 (to be precise, sequence number 57)), a search was performed using the BLAST server provided by NCBI, and the result was that there was no amino acid sequence with a sequence identity of more than 97% for either. The amino acid sequence with the highest sequence identity of about 96% is the amino acid sequence of the tail tube protein of ΦXc10 and f20-Xaj mentioned above.

[0610] Based on the above suggestions, these proteins are new tail tube proteins that mediate bacteriolysis against Xanthomonas bacteria.

[0611] <Example 8: Isolation of a new bacteriophage and its bacteriolytic activity (8)>

[0612] (Purpose)

[0613] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0614] (Methods and Results)

[0615] (1) Acquisition and cultivation of plant pathogenic bacteria

[0616] In the present invention, the plant pathogenic bacteria used as target bacteria are Xanthomonas bacteria, and all strains are obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 11 together with their respective NARO accession numbers (MAFF No.).

[0617] [Table 11]

[0618] ID Species name Separation source Collection location MAFF No.211971 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.311282 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.301420 Xanthomonas arboricola pv.pruni Peach Nagano MAFF No.301426 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311351 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311414 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311417 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311562 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.311571 Xanthomonas arboricola pv.pruni Peach Fukushima MAFF No.311618 Xanthomonas arboricola pv.pruni Peach Wakayama MAFF No.311622 Xanthomonas arboricola pv.pruni Peach Yamanashi NCIMB-ID: 10460 Pseudomonas fluorescens egg USA

[0619] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0620] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0621] (2) Isolation and purification of phage 8

[0622] New bacteriophages were isolated from natural sewage, soil, or plant bodies (leaves) obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, five new bacteriophages were isolated from natural sewage / soil or water obtained by immersing plant bodies. The new bacteriophage isolated in Example 8 is referred to as "the eighth bacteriophage."

[0623] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0624] (3) Amplification and purification of phage 8

[0625] In order to amplify and purify the isolated and purified 8th phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the 1st phage" of Example 1. The titer of the prepared 8th phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0626] (4) Evaluation of the host range of phage 8

[0627] The host range of the eighth phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0628] An example of the results is shown in Figure 8 and Fig. 9 . When the 8th phage showed lytic activity, in the bacterial moss formed on the plate, only the part to which the phage purification solution was dropped became transparent. The 8th phage obtained by the present invention showed lytic activity against all Xanthomonas bacteria shown in Table 11. On the other hand, it did not show lytic activity against Pseudomonas fluorescens. As in Example 3, lytic activity was shown against any Xanthomonas bacteria isolated from different regions. This suggests that the isolated 8th phage showed lytic activity against a wide range of Xanthomonas bacteria.

[0629] In addition, these results suggest that phage 8 is useful for controlling peach bacterial borer disease caused by Xanthomonas bacteria.

[0630] (5) Genome analysis of phage 8

[0631] The genomic DNA sequence of phage No. 8 was determined and analyzed.

[0632] (i) Preparation and sequence determination of genomic DNA of phage No. 8

[0633] Using TURBO DNA-free TM The genome of the eighth phage was extracted using a kit (Thermo Fisher Scientific). The basic operation was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0634] (ii) Bioinformatics analysis based on genome sequence information

[0635] Based on the genomic base sequence of the 8th phage obtained in (i) above (sequence numbers 18 and 19), a search for similar DNA sequences and confirmation of sequence identity were performed using the BLAST server provided by NCBI (same as above). As a result of the search, there was no sequence with a sequence identity of 80% or more to any of the base sequences in sequence numbers 32 to 36 within a range of more than 50% of the total length. In addition, there was no sequence with a sequence identity of 90% or more to any of the base sequences in sequence numbers 32 to 36 within a range of more than 20% of the total length. This result suggests that the 8th phage isolated in Example 8 is basically a phage with a new genomic sequence for which there is no reported example of a related sequence.

[0636] In addition, the sequence identities between the genomic DNA sequences of each phage of sequence number 32 to 36 were subjected to BLAST analysis using GENETYX-NGS installed in the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ). The sequence identities using the average nucleotide identity (ANI) as an indicator are shown in Table 12 below.

[0637] [Table 12]

[0638] Sequence number 32 Sequence Number 33 Sequence Number 34 Sequence Number 35 Sequence Number 36 Sequence number 32 99.98 / 99.33 98.41 / 98.96 100 / 99.86 99.97 / 97.57 Sequence Number 33 99.98 / 97.72 98.47 / 99.03 99.97 / 99.79 99.91 / 97.79 Sequence Number 34 99.52 / 93.22 99.55 / 93.12 99.52 / 90.98 99.54 / 90.83 Sequence Number 35 100 / 97.70 99.97 / 97.70 99.15 / 94.81 99.89 / 99.65 Sequence Number 36 99.88 / 99.70 99.91 / 99.69 99.44 / 93.11 99.88 / 97.72

[0639] In Table 12, the ANI value and comparison range of the genomic DNA sequence of each row relative to the genomic DNA sequence of each column are expressed in the form of "ANI value (%) / comparison range (%)". It should be noted that the comparison range (%) in Table 12 is the ratio of the region in the genomic DNA sequence of the corresponding column that is matched to the genomic DNA sequence of the corresponding row. For example, when the ANI value (%) / comparison range (%) is "90 / 90", the genomic DNA sequence of the corresponding row has a sequence identity of 90% with respect to the genomic DNA sequence of the corresponding column within 90% of the total length.

[0640] Since the lengths of the genomic DNA sequences of the phages of sequence numbers 32 to 36 are different, the ANI values ​​and comparison ranges of the genomic DNA sequences of each row in Table 12 relative to the genomic DNA sequences of each column are sometimes slightly different from the ANI values ​​and comparison ranges when the row and column are swapped. For example, when the ANI values ​​and comparison ranges of the row of sequence number 32 and the column of sequence number 35 are compared with the ANI values ​​and comparison ranges of the row of sequence number 35 and the column of sequence number 32, although the ANI values ​​of both are 100%, the values ​​of the comparison ranges are different because the lengths of the genomes of sequence numbers 32 and 35 are different.

[0641] As shown in Table 12, the base sequences of sequence numbers 32 to 36 have a sequence identity of 98 to 100% in the range of 90% to 100%, and it was found that they are very similar to each other. The ANI value and the comparison range in Table 12 were multiplied to estimate the sequence identity of the entire range. The result was that the sequence identity of sequence number 35 to sequence number 36 was the lowest in the entire range, which was 90%.

[0642] The results of comparison of the genomic DNA sequences of the above-mentioned phages suggest that a phage having a genomic DNA sequence comprising a base sequence having a sequence identity of 90% or more with any of the base sequences in sequence numbers 32 to 36, that is, a phage having a genomic DNA sequence comprising a base sequence in which about 10% of the bases are added, deleted and / or substituted in any of the base sequences in sequence numbers 32 to 36, essentially also has the same host range / lytic activity as a phage having a genomic DNA sequence in any of the base sequences in sequence numbers 32 to 36.

[0643] <Example 9: Isolation of a new bacteriophage and its bacteriolytic activity (9)>

[0644] (Purpose)

[0645] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0646] (Methods and Results)

[0647] (1) Acquisition and cultivation of plant pathogenic bacteria

[0648] In the present invention, the target plant pathogenic bacteria are Xanthomonas bacteria, and all strains are obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are listed in Table 13 together with their respective NARO accession numbers (MAFF No.).

[0649] [Table 13]

[0650] ID Species name Separation source Collection location MAFF No.311622 Xanthomonas arboricola pv.pruni Peach Yamanashi MAFF No.212146 Xanthomonas arboricola pv.juglandis Walnut Nagano MAFF No.301078 Xanthomonas citri subsp. citri Orange Wakayama MAFF No.301260 Xanthomonas campestris pv.vesicatoria chili Tokushima MAFF No.301352 Xanthomonas campestris pv.vitians lettuce Wakayama MAFF No.301354 Xanthomonas campestris pv.vitians lettuce Chiba MAFF No.106783 Xanthomonas campestris pv.campestris Chinese cabbage Shizuoka MAFF No.106181 Xanthomonas campestris pv.raphani radish Aomori NCIMB-ID 10460 Pseudomonas fluorescens egg USA

[0651] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) which is beneficial to the environment and has been reported to have plant growth promoting effects and the like was obtained from NCIMB, a research institute within UKNCC, a British microbial strain collection.

[0652] The culture of various Xanthomonas bacteria and Pseudomonas fluorescens was carried out according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" of Example 1.

[0653] (2) Isolation and purification of phage 9

[0654] New bacteriophages were isolated from natural sewage, soil, or plant bodies (leaves) obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, five new bacteriophages were isolated from natural sewage / soil or water obtained by immersing plant bodies. The new bacteriophage isolated in Example 9 is referred to as "9th bacteriophage".

[0655] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0656] (3) Amplification and purification of phage No. 9

[0657] In order to amplify and purify the separated and purified 9th phage, the plate lysate (PL) method was implemented. The specific method was based on the method described in "(3) Amplification and purification of the 1st phage" of Example 1. The titer of the prepared 9th phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0658] (4) Evaluation of the host range of phage 9

[0659] The host range of the ninth phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0660] An example of the results is shown in Fig.10. When the phage shows lytic activity, in the bacterial moss formed on the plate, only the part where the phage purification solution was dropped becomes transparent. The phage obtained by the present invention shows lytic activity against all strains belonging to the three species of Xanthomonas arboricola, Xanthomonas citri and Xanthomonas campestris shown in Table 13. In addition, for Xanthomonas arboricola and Xanthomonas campestris, lytic activity was also shown against strains of different pathogenic types. On the other hand, for Pseudomonasfluorescens, it was also confirmed that no lytic activity was shown. Although there are reports of phages showing activity against more than two species of Xanthomonas, such a combination including the pathogenic type has not been reported (Nakayinga R. et al., BMC Microbiology, 2021, 21: 291). This is a result showing the possibility that the phage of the present invention can be applied to various plant diseases. For example, it is suggested that the compound is useful for controlling peach bacterial scald, citrus canker, cauliflower black rot, and the like, which are caused by Xanthomonas bacteria, and is expected to have high value in industrial applications.

[0661] (5) Genome analysis of phage 9

[0662] The genomic DNA sequence of phage No. 9 was determined and analyzed.

[0663] (i) Preparation and sequence determination of genomic DNA of phage No. 9

[0664] Using TURBO DNA-free TM Kit (Thermo Fisher Scientific) was used to extract the genome of the 9th phage. The basic operation was based on the method described in Example 1, "(5) Analysis of the genome of the 1st phage (i) Preparation and sequence determination of the 1st phage genomic DNA".

[0665] (ii) Bioinformatics analysis based on genome sequence information

[0666] Based on the genome base sequence (sequence number 41) of the 9th phage obtained in (i) above, the BLAST server provided by NCBI (same as above) was used to implement the search for similar DNA sequences and confirmation of sequence identity. The result of the search was that the genome sequence of the phage "Xanthomonas phage Xaa_vBphi31" (NCBI accession number: MT951568.1) included in the NCBI database had the highest affinity score. It has about 85% sequence identity within about 80% of the total length. According to this result, it can be inferred that the sequence identity relative to the total length is equivalent to a value of about 68%. As the host of Xaa_vB_phi31, only Xanthomonas euvesicatoria pv.allii XaaBL11 is recorded. For other phages with high affinity scores, there is no information suggesting that the 9th phage widely shows lytic activity against various species of Xanthomonas bacteria.

[0667] In order to explore the cause of the difference in target bacteria, a search for new genes was conducted in the genome base sequence information of the 9th phage (sequence number 41). The gene region was estimated using the RAST server (http: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ). As a result of the search, a gene group encoding a new tail tubular protein was identified as a gene related to the host range of the phage.

[0668] Sequence number 39 represents the base sequence of the tail tube protein A gene contained in the genome base sequence (sequence number 41) of the 9th phage discovered in this example, and sequence number 37 represents the amino acid sequence of the tail tube protein A. On the other hand, sequence number 40 represents the base sequence of the tail tube protein B gene, and sequence number 38 represents the amino acid sequence of the tail tube protein B.

[0669] Since tail tube protein A and tail tube protein B have been reported to be associated with the specific adsorption of phages to bacteria (Maozhi Hu, et ai., 2020, 9: 1, 855-867), the possibility of being associated with the host range of the phage is also high for the 9th phage. However, based on the amino acid sequence of tail tube protein A (sequence number 37) and the amino acid sequence of tail tube protein B (sequence number 38), a search was performed using the BLAST server provided by NCBI, and the result was that there was no amino acid sequence with a sequence identity of more than 97% for either. Relative to the amino acid sequence of tail tube protein A, the amino acid sequence of tail tube protein A of the above-mentioned Xanthomonas phage Xaa_vB_phi31 showed the highest sequence identity of about 95%. The sequence identity of the base sequence of sequence number 39 to the base sequence of tail tube protein A encoding Xaa_vB_phi31 is about 89%. The amino acid sequence of the tail tube protein B of Xaa_vB_phi31 showed the highest sequence identity of about 91% with respect to the amino acid sequence of the tail tube protein B. The base sequence of this gene also showed a sequence identity of about 86% with the base sequence of SEQ ID NO: 40.

[0670] Based on the above suggestions, these proteins are new tail tube proteins that mediate bacteriolysis against Xanthomonas bacteria.

[0671] <Example 10: Isolation of a new bacteriophage and its bacteriolytic activity (10)>

[0672] (Purpose)

[0673] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0674] (Methods and Results)

[0675] (1) Acquisition and cultivation of plant pathogenic bacteria

[0676] In this example, the plant pathogenic bacteria were Xanthomonas bacteria, and all strains were obtained from the Agricultural Research Organization (NARO). The bacteria used in this example are the same as those listed in Table 13 of Example 9.

[0677] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0678] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0679] (2) Isolation and purification of phage 10

[0680] A new bacteriophage was isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, a new bacteriophage was isolated from natural sewage / soil. The new bacteriophage isolated in Example 10 is referred to as "the tenth bacteriophage".

[0681] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0682] (3) Amplification and purification of phage 10

[0683] In order to amplify and purify the isolated and purified phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the first phage" of Example 1. The titer of the 10th phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0684] (4) Evaluation of the host range of phage 10

[0685] The host range of the tenth phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0686] An example of the results is shown in Fig.11. When the phage shows lytic activity, in the bacterial moss formed on the plate, only the part where the phage purification solution was dropped becomes transparent. The 10th phage shows lytic activity against all strains belonging to the three species of Xanthomonas arboricola, Xanthomonas citri and Xanthomon as campestris shown in Table 13. In addition, for Xanthom onas arboricola and Xanthomonas campestris, lytic activity was also shown against strains of different pathotypes. On the other hand, it was also confirmed that no lytic activity was shown for Pseudomonas fluorescens. Although there are reports of phages showing activity against more than two species of Xanthomonas, such a combination including pathotypes has not been reported (Nakayinga R. et al., BMC Microbiology, 2021, 21: 291). This is a result showing the possibility that the 10th phage can be applied to various plant diseases. For example, it is suggested that the compound is useful for controlling peach bacterial scald, citrus canker, cauliflower black rot, and the like, which are caused by Xanthomonas bacteria, and is expected to have high value in industrial applications.

[0687] (5) Genome analysis of phage 10

[0688] The genomic DNA sequence of the 10th phage was determined and analyzed.

[0689] (i) Preparation and sequence determination of genomic DNA of phage 10

[0690] Using TURBO DNA-free TM Kit (Thermo Fisher Scientific, Inc.) was used to extract the genome of the tenth phage. The basic operation was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage."

[0691] (ii) Bioinformatics analysis based on genome sequence information

[0692] Based on the genomic base sequence (sequence number 44) of the 10th phage obtained in (i) above, the BLAST server provided by NCBI (same as above) was used to implement the search for similar DNA sequences and confirmation of sequence identity. The result of the search is that the phage isolated in the United States (name: RiverRider; NCBI accession number: NC_048703.1) recorded in the document of Miller et al. (Miller M.et al., Archives of Viro logy, 2020, 165: 1481-1484) has the highest kinship score. Relative to sequence number 44, the sequence identity is about 91% within the range of about 80% of the total length, and the sequence identity in the full length of the genomic sequence is less than 80% (approximately equivalent to 73%). According to the result, the 10th phage isolated in this embodiment is a phage with a new genomic sequence.

[0693] Generally, regarding the host specificity and host range characteristics of bacteriophages, the role of the tail fiber protein is considered to be very important (Nobrega F. Let al., Nat. Rev. Microbiol., 2018, 16: 760-773). Therefore, the tail fiber gene was retrieved from the genome sequence information of the above-mentioned bacteriophage. The base sequence shown in sequence number 43, which is presumed to be the tail fiber gene, was determined from the genome sequence of the 10th phage using the RAST server (https: / / rast.nmpdr.org / ) and PHASTER (https: / / phaster.ca / ). A search for similar DNA sequences was conducted for this gene sequence, and the result was that the sequence of the tail fiber gene of the above-mentioned bacteriophage RiverRider had the highest affinity score. The sequence identity of this base sequence is about 77% in the first half of the base sequence of sequence number 43 (1st to 890th positions: equivalent to about 52% of the total length). Regarding the amino acid sequence of sequence number 42, the highest affinity score is the tail fiber protein of RiverRider. The sequence identity of the amino acid sequence is about 82% within about 52% of the total length. It can be seen from this that the tail fiber protein of the 10th phage is a new tail fiber protein that does not have a high affinity protein, and is a particularly characteristic protein in the phage of the present invention compared to known phages.

[0694] Regarding the host bacteria of the phage RiverRider, the above document records that it exhibits lytic activity against Xanthomonas fragariae, but does not exhibit lytic activity against Xanthomonas arboricola and Xanthomonas cam pestris. Therefore, the host range is significantly different from that of the 10th phage, suggesting that the huge difference in the host range between the phage RiverRider and the phage obtained in this example is caused by the difference in the tail fiber gene.

[0695] Based on the above results, it is estimated that the tenth phage has a new host range of Xanthomonas that has not been reported before, and its characteristics are particularly based on the novel tail fiber gene.

[0696] <Example 11: Isolation of a novel second phage and its bacteriolytic activity>

[0697] (Purpose)

[0698] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0699] (Methods and Results)

[0700] (1) Acquisition and cultivation of plant pathogenic bacteria

[0701] In this example, the plant pathogenic bacteria used as the target bacteria were Xanthomonas bacteria, and all strains were obtained from the Agricultural Research Organization (NARO). In this example, in addition to the bacteria listed in Table 4, the bacteria listed in Table 14 were also used.

[0702] [Table 14]

[0703] ID Species name Separation source Collection location MAFF No.106765 Xanthomonas campestris pv.campestris brocoli Ibaraki

[0704] In addition, as a control, a strain of Pseudomonas fluorescens (NCIM B-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0705] Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0706] (2) Isolation and purification of bacteriophage

[0707] New bacteriophages were isolated from natural sewage or soil obtained in Japan. The isolation and purification methods were based on the methods described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, two new bacteriophages were isolated from natural sewage / soil.

[0708] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0709] (3) Phage amplification and purification

[0710] In order to amplify and purify the isolated and purified phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the first phage" of Example 1. The titer of the prepared phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0711] (4) Evaluation of the host range of bacteriophages

[0712] The host range of the phage was evaluated by the spot test method. The basic operation was based on the method described in "(4) Evaluation of the host range of the first phage" in Example 1.

[0713] An example of the results is shown in Fig.14 and 15 When the phage showed lytic activity, only the part where the phage purification solution was dropped in the bacterial lawn formed on the plate became transparent. The two phages obtained by the present invention showed lytic activity against all Xanthomonas bacteria shown in Table 4, similarly to the second phage ( Fig.14 ). On the other hand, no lytic activity was shown against Pseudomonas fluorescens. This suggests that the two isolated phages may have a high affinity with the second phage. In addition, the second phage obtained in Example 2 and the two phages obtained in this example confirmed lytic activity against Xanthomonas campestris pv. Campestris. As a result, all three phages showed lytic activity against this strain ( Fig.15 ).

[0714] In addition, this result suggests that the phage obtained in this Example is useful for controlling peach bacterial blowhole, rice bacterial blight, citrus canker, cauliflower black rot, and the like, which are caused by Xanthomonas bacteria.

[0715] (5) Phage genome analysis

[0716] The genomic DNA sequence of the phage obtained in this example was determined and analyzed.

[0717] (i) Preparation and sequence determination of phage genomic DNA

[0718] Using TURBO DNA-free TM The genomes of the two phages were extracted using a kit (Thermo Fisher Scientific). The basic procedure was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0719] (ii) Bioinformatics analysis based on genome sequence information

[0720] Based on the genomic base sequences (SEQ ID NOs. 47 and 48) of the phages obtained in (i) above, the average nucleotide identity (ANI) with respect to the genomic base sequence (SEQ ID NO. 13) of the second phage was analyzed using GENETYX-NGS. As a result of the analysis, the average nucleotide identity (AM) with respect to SEQ ID NO. 13 was 98.90% for SEQ ID NO. 47 and 98.92% for SEQ ID NO. 48. Therefore, the two phages obtained in this example and the phages obtained in Example 2 are also collectively referred to as "second phages".

[0721] In order to clarify the reason for the identical host specificity between the phage isolated in this example and the phage isolated in Example 2, the sequences of the fiber proteins of the two were compared.

[0722] First, the tail fiber gene was 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 gene. As a result, the base sequence shown in sequence number 46 was determined as the base sequence of the tail fiber gene. The base sequences of the tail fiber genes of the two phages isolated in this example are the same.

[0723] Furthermore, the amino acid sequence (SEQ ID NO. 45) encoded by the gene containing the base 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 bacteriophage isolated in Example 2. As a result, only the type of amino acid at position 154 was different. Specifically, the threonine (Thr) at position 154 in SEQ ID NO. 11 was changed to alanine (Ala).

[0724] In relation to the phage Xp12 having the highest affinity score with the phage isolated in Example 2, relative to the amino acid sequence of its affinity protein, in addition to the differences in the types of amino acids at positions 278 and 350 as in the phage isolated in Example 2, the type of the amino acid at position 154 mentioned above is also different in the phage obtained in this Example.

[0725] As described in Example 2, it is known that the only bacterial species that phage Xp12 exhibits lytic activity is Xanthomonas oryzae, and therefore, it is suggested that the lytic activity of the second phage against a wide range of hosts is caused by the difference between the amino acids at positions 278 and 350 in the amino acid sequence of the fiber protein. In addition, it is suggested that even a phage having different amino acids at other positions by addition, such as the second phage obtained in this Example, has a wide host range.

[0726] <Example 12: Isolation of novel phage 7 and its bacteriolytic activity>

[0727] (Purpose)

[0728] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0729] (Methods and Results)

[0730] (1) Acquisition and cultivation of plant pathogenic bacteria

[0731] In the present invention, the plant pathogenic bacteria used as the target bacteria are Xanthomonas bacteria, and all strains are obtained from the Agricultural Research Organization (NARO). In addition to the Xanthomonas bacteria shown in Table 10 used in Example 7, the bacteria used in this example are listed in Table 15 together with the accession number (MAFF No.) of the Agricultural Research Organization.

[0732] [Table 15]

[0733] ID Species name Separation source Collection location MAFF No.301260 Xanthomonas campestris pv.vesicatoria chili Tokushima

[0734] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British collection of microbial strains.

[0735] The culture of Xanthomonas bacteria and Pseudomonas fluorescens was carried out according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" of Example 1.

[0736] (2) Isolation and purification of bacteriophage

[0737] A new bacteriophage was isolated from natural sewage or soil obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, a new bacteriophage was isolated from natural sewage / soil.

[0738] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0739] (3) Phage amplification and purification

[0740] In order to amplify and purify the isolated and purified phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the first phage" of Example 1. The titer of the prepared phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0741] (4) Evaluation of the host range of bacteriophages

[0742] The host range of the obtained phage was evaluated. The basic operation was based on the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0743] An example of the results is shown in Fig.16 and 17 When the phage showed lytic activity, only the part where the phage purification solution was dropped in the bacterial lawn formed on the plate became transparent. The phage obtained by the present invention showed lytic activity against all Xanthomonas bacteria shown in Table 10 ( Fig.16 ). On the other hand, no lytic activity was shown against Pseudomonas fluorescens. This suggests that the isolated phage may have a high affinity with the seventh phage. In addition, the two seventh phages obtained in Example 7 and the phages obtained in this example confirmed lytic activity against Xanthomonas campestris pv. Vesicatoria. As a result, all three phages showed lytic activity against this strain ( Fig.17 ).

[0744] In addition, this result suggests that the bacteriophage obtained in this Example is useful for controlling peach bacterial borer disease and cauliflower black rot disease caused by Xanthomonas bacteria.

[0745] (5) Genome analysis of phage 7

[0746] The genomic DNA sequence of the phage obtained in this example was determined and analyzed.

[0747] (i) Preparation and sequence determination of phage genomic DNA

[0748] Using TURBO DNA-free TM The phage genome was extracted using a kit (Thermo Fisher Scientific). The basic procedure was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0749] (ii) Bioinformatics analysis based on genome sequence information

[0750] Based on the genomic base sequence of the phage obtained in (i) above (sequence number 53), the average nucleotide identity (ANI) relative to the genomic base sequence of the seventh phage (sequence numbers 28 and 29) was analyzed using GENE TYX-NGS. As a result, the base sequence shown in sequence number 53 has a sequence identity of 92.61% within a range of 75.06% with respect to the base sequence shown in sequence number 28. On the other hand, with respect to the base sequence shown in sequence number 29, it has a sequence identity of 92.19% within a range of 77.05%. The identity of the genomic sequence is not very high, which is unexpected.

[0751] In order to clarify the reason for the identical host specificity between the phage isolated in this example and the phage isolated in Example 7, the sequences of the tail tube proteins of the two were compared.

[0752] First, the gene group encoding the tail tube protein was identified from the genome sequence of the bacteriophage of this example. The genes were identified using the BRAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ).

[0753] Sequence number 51 represents the base sequence of the tail tube protein A gene contained in the genome base sequence (sequence number 53) of the bacteriophage discovered in this example, and sequence number 49 represents the amino acid sequence of the tail tube protein A. On the other hand, sequence number 52 represents the base sequence of the tail tube protein B gene, and sequence number 50 represents the amino acid sequence of the tail tube protein B.

[0754] Furthermore, the amino acid sequences of these tail tube proteins (SEQ ID NOs: 49 and 50) were compared with the amino acid sequences of the tail tube proteins of the phage isolated in Example 7 (SEQ ID NOs: 24 and 58). As a result, the amino acid sequence of the tail tube protein A had a sequence identity of 96.58%, and the amino acid sequence of the tail tube protein B had a sequence identity of 97.60%. Therefore, the phage obtained in this example and the phage obtained in Example 7 are also collectively referred to as "the seventh phage".

[0755] Specifically, regarding the amino acid sequence of tail tube protein A, in the amino acid sequence of sequence number 49, the following seven amino acid substitutions were found based on the amino acid sequence shown in sequence number 24: substitution of glutamic acid (Glu) at position 28 for aspartic acid (Asp), substitution of serine (Ser) at position 108 for asparagine (Asn), substitution of glutamine (Gln) at position 110 for histidine (His), substitution of glutamine (Gln) at position 153 for lysine (Lys), substitution of aspartic acid (Asp) at position 157 for asparagine (Asn), substitution of tyrosine (Tyr) at position 189 for phenylalanine (Phe), and substitution of valine (Val) at position 201 for tyrosine (Tyr).

[0756] On the other hand, regarding the amino acid sequence of tail tube protein B, in the amino acid sequence of SEQ ID NO: 50, the following 20 amino acid substitutions were found based on the amino acid sequence shown in SEQ ID NO: 58: substitution of alanine (Ala) at position 25 to proline (Pro), substitution of alanine (Ala) at position 53 to serine (Se r), substitution of alanine (Ala) at position 216 for proline (Pro), substitution of histidine (His) at position 221 for tyrosine (Tyr), substitution of valine (Val) at position 272 for (Ile), substitution of alanine (Ala) at position 389 for glutamic acid (Glu), substitution of serine (Ser) at position 395 for alanine (Ala), substitution of valine (Val) at position 410 for isoleucine (Ile), substitution of isoleucine (Ile) at position 425 for valine (Val), substitution of glycine (Gly) at position 472 for alanine (Ala), substitution of alanine (Ala) at position 607 for serine (Ser), The substitution of isoleucine (Ile) at position 623 with valine (Val), the substitution of arginine (Arg) at position 662 with serine (Ser), the substitution of leucine (Leu) at position 670 with glutamine (Gln), the substitution of threonine (Thr) at position 699 with asparagine (Asn), the substitution of aspartic acid (Asp) at position 707 with glycine (Gly), the substitution of serine (Ser) at position 757 with alanine (Ala), the substitution of glycine (Gly) at position 763 with serine (Ser), the substitution of serine (Ser) at position 764 with asparagine (Asn), and the substitution of methionine (Met) at position 765 with valine (Val).

[0757] The relationship with the phages ΦXc10 and f20-Xaj having the highest affinity score with respect to the amino acid sequence of the tail tube protein of the phage isolated in Example 7 was investigated. As a result, regarding the tail tube protein A, SEQ ID NOs: 24 and 49 belong to the amino acid sequence shown in SEQ ID NO: 60, whereas there are no known phages including ΦXc10 and f20-Xaj that belong to the amino acid sequence shown in SEQ ID NO: 60. In addition, regarding the tail tube protein B, SEQ ID NOs: 58 and 50 belong to the amino acid sequence shown in SEQ ID NO: 61, whereas there are no known phages including ΦXc10 and f20-Xaj that belong to the amino acid sequence shown in SEQ ID NO: 61.

[0758] As described in Example 7, phages ΦXc10 and f20-Xaj, etc., do not show lytic activity against Xanthomonas arboricola pv. pruni, etc., and therefore, it is suggested that the lytic activity unique to the seventh phage is caused by the difference in the amino acid sequence of the tail tube protein. In addition, it is suggested that even when there is a certain degree of difference in the amino acid sequence of the tail tube protein as in the seventh phage obtained in this example, it is suggested that the host range is the same depending on the position.

[0759] <Example 13: Isolation of novel 9th ​​phage and its bacteriolytic activity>

[0760] (Purpose)

[0761] A new bacteriophage having lytic activity against pathogenic bacteria of plant diseases is isolated, and its lytic activity against plant pathogenic bacteria is verified.

[0762] (Methods and Results)

[0763] (1) Acquisition and cultivation of plant pathogenic bacteria

[0764] In the present invention, the plant pathogenic bacteria as the target bacteria are Xanthomonas bacteria, and all strains are obtained from the Agricultural Research Organization (NARO). In this example, the Xanthomonas bacteria listed in Table 13 were used.

[0765] As a control, a strain of Pseudomonas fluorescens (NCIMB-ID: 10460) was obtained from NCIMB, a research institute within UKNCC, a British microbial strain collection, which has been reported to have plant growth-promoting effects and other environmental benefits.

[0766] The culture of various Xanthomonas bacteria and Pseudomonas fluorescens was carried out according to the method described in "(1) Acquisition and culture of plant pathogenic bacteria" in Example 1.

[0767] (2) Isolation and purification of bacteriophage

[0768] A novel bacteriophage was isolated from natural sewage, soil, or plant (leaves) obtained in Japan. The isolation method and purification method were based on the method described in "(2) Isolation and purification of the first bacteriophage" of Example 1. As a result, a novel bacteriophage was isolated from natural sewage / soil or water obtained by immersing plant bodies.

[0769] The separated phages were recovered as a phage-containing liquid suspended in SM Buffer and passed through a 0.2 μm filter. The phage-containing liquid was mixed with the bacterial solution under the above conditions to separate the phages again. The phages were purified by repeating this step several times.

[0770] (3) Phage amplification and purification

[0771] In order to amplify and purify the isolated and purified phage, the plate lysate (PL) method was performed. The specific method was based on the method described in "(3) Amplification and purification of the first phage" of Example 1. The titer of the prepared phage purified solution was determined by plaque assay using a properly diluted solution and was confirmed to be 10 8 PFU / mL or above.

[0772] (4) Evaluation of the host range of bacteriophages

[0773] The host range of the obtained phage was evaluated. The basic operation was based on the method described in "(4) Evaluation of host range of the first phage" in Example 1.

[0774] An example of the results is shown in Fig.18 When the phage showed lytic activity, only the part of the bacterial lawn formed on the plate where the phage purification solution was dropped became transparent. The phage obtained in this example showed lytic activity against all strains belonging to the three species of Xanthomonas arboricola, Xanthomonas citri and Xanthomonas campestris, similarly to the phage obtained in Example 9 ( Fig.18 ). In addition, it was confirmed that the phage showed lytic activity against strains of different pathogenic types. On the other hand, it was also confirmed that the phage showed no lytic activity against Pseudomonas fluorescens. This suggests that the isolated phage may have a high affinity with the ninth phage.

[0775] In addition, this result suggests that the bacteriophage obtained in this Example is useful for controlling, for example, peach bacterial scald, citrus canker, and cauliflower black rot caused by Xanthomonas bacteria.

[0776] (5) Phage genome analysis

[0777] The genomic DNA sequence of the obtained phage was determined and analyzed.

[0778] (i) Preparation and sequence determination of phage genomic DNA

[0779] Using TURBO DNA-freeTM The phage genome was extracted using a kit (Thermo Fisher Scientific). The basic procedure was based on the method described in Example 1, "(5) Analysis of the genome of the first phage (i) Preparation and sequence determination of the genomic DNA of the first phage".

[0780] (ii) Bioinformatics analysis based on genome sequence information

[0781] Based on the genomic base sequence of the phage obtained in (i) above (SEQ ID NO: 56), the average nucleotide identity (ANI) with respect to the genomic base sequence of the 9th phage (SEQ ID NO: 41) was analyzed using GENE TYX-NGS. As a result, the base sequence shown in SEQ ID NO: 56 had a sequence identity of 92.61% within a range of 75.06% with respect to the base sequence shown in SEQ ID NO: 41. It was unexpected that the identity of the genomic sequence was not very high.

[0782] In order to clarify the reason for the identical host specificity between the phage isolated in this example and the phage isolated in Example 9, the sequences of the tail tube proteins of the two were compared.

[0783] First, the gene group encoding the tail tube protein was identified from the genome sequence of the bacteriophage of this example. The genes were identified using the BRAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ).

[0784] The base sequence of the tail tube protein A gene contained in the genome base sequence (SEQ ID NO: 56) of the phage found in this example is completely identical to the base sequence (SEQ ID NO: 39) of the tail tube protein A gene of the phage isolated in Example 9. Therefore, the amino acid sequence of the tail tube protein A obtained by translation is identical to the amino acid sequence (SEQ ID NO: 37) of the tail tube protein A of the phage isolated in Example 9. Therefore, the phage obtained in this example and the phage obtained in Example 9 are also collectively referred to as "9th phage".

[0785] On the other hand, SEQ ID NO: 55 shows the base sequence of the tail tube protein B gene, and SEQ ID NO: 54 shows the amino acid sequence of the tail tube protein B.

[0786] Furthermore, the amino acid sequence of the tail tube protein B (SEQ ID NO: 54) was compared with the amino acid sequence of the tail tube protein B of the bacteriophage isolated in Example 9 (SEQ ID NO: 38). The sequence identity of the amino acid sequence of the tail tube protein B was 99.3%. Specifically, in the amino acid sequence shown in SEQ ID NO: 54, the following six amino acid substitutions were found based on the amino acid sequence shown in SEQ ID NO: 38: substitution of threonine (Thr) at position 61 with alanine (Ala), substitution of glutamine (Gln) at position 108 with lysine (Lys), substitution of proline (Pro) at position 404 with glutamine (Gln), substitution of proline (Pro) at position 679 with serine (Ser), substitution of threonine (Thr) at position 682 with alanine (Ala), and substitution of isoleucine (Ile) at position 727 with valine (Val).

[0787] As described in Example 9, there is no known phage that has a sequence identity of 97% or more with respect to the amino acid sequences of the tail tube proteins A and B of the 9th phage obtained in Example 9. In addition, it is not known that the phage Xanthomonas phage Xaa_vB_phi31 and the like, which have a high affinity score with the 9th phage, widely exhibit lytic activity against various species of the 9th phage Xanthomonas bacteria. Thus, it is suggested that the wide lytic activity of the 9th phage is caused by the difference in the amino acid sequence of the tail tube protein. In addition, it is suggested that even in the case where there is a certain degree of difference in the amino acid sequence of the tail tube protein, such as the 9th phage obtained in this example, the same host range is present.

[0788] <Example 14: Lytic activity of the obtained phage combination>

[0789] (Purpose)

[0790] New bacteriophages that have shown lytic activity against pathogenic bacteria of plant diseases when used alone were examined for their effects on plant diseases when used in combination.

[0791] (method)

[0792] (1) Spot test method

[0793] The spot test was performed according to the method described in "(4) Evaluation of host range of first phage" of Example 1. In this example, the strain Xanthomonas arboricol pv. pruni (MAFF No. 311351) was used, in which lytic activity was confirmed for all phages tested.

[0794] As the phage purified solution to be added dropwise, a mixed solution prepared by mixing equal amounts of the phage purified solutions prepared in each example was used. The mixed solution was prepared by appropriately diluting the total titer to the same degree as when used alone. The phage used in this example is as follows.

[0795] In this embodiment, the following phages were used: a phage having a genomic DNA sequence of sequence number 8 as the first phage, a phage having a genomic DNA sequence of sequence number 13 as the second phage, a phage having a genomic DNA sequence of sequence number 14 as the third phage, a phage having a genomic DNA sequence of sequence number 17 as the fourth phage, a phage having a genomic DNA sequence of sequence number 18 as the fifth phage, a phage having a genomic DNA sequence of sequence number 23 as the sixth phage, a phage having a genomic DNA sequence of sequence number 28 as the seventh phage, a phage having a genomic DNA sequence of sequence number 32 as the eighth phage, a phage having a genomic DNA sequence of sequence number 41 as the ninth phage, and a phage having a genomic DNA sequence of sequence number 44 as the tenth phage.

[0796] (result)

[0797] An example of the results is shown in Fig.19 and 20 When the combination of phages showed lytic activity, only the part of the bacterial lawn formed on the plate where the phage purification solution was dropped became transparent. It can be seen that when any combination of 2, 5, and 8 types of phages was used, lytic activity was at least the same as when each phage was used alone ( Fig.19 and 20 ).

[0798] From the above results, it was confirmed that the bacteriophages of the present invention are useful whether used alone or in combination.

[0799] <Example 15: Disease control effect test of peach bacterial puncture>

[0800] (Purpose)

[0801] The effects of isolated new bacteriophages having lytic activity against pathogenic bacteria of plant diseases on plant diseases when applied to plants were verified.

[0802] (method)

[0803] (1) Preparation of phage dissemination fluid

[0804] The phage dispersion solution used in this experiment was prepared according to the following procedure.

[0805] First, a bacterial culture solution of a host bacterium for phage amplification was prepared according to the following steps. As a host, the strain Xanthomon as arboricolapv.pruni (MAFF No. 311351) was used, which was confirmed to have lytic activity among all the phages tested. The bacterial cells were inoculated into YPG Broth and incubated overnight in a shaker set at 25°C. After incubation, the OD was measured. 600 (turbidity at a wavelength of 600 nm), and the culture solution that reaches about 1.0 is used as a bacterial culture solution.

[0806] The prepared bacterial culture solution was mixed with the purified phage solution containing one phage prepared in Examples 1 to 13 (titer of 10 8 PFU / mL) were mixed in equal amounts, the resulting mixture was inoculated into 100 times the amount of YPG culture solution, incubated for about 8 to 12 hours in a shaker set at 25°C, and the resulting culture solution was recovered as a phage crude solution. 1 / 10 of the recovered crude solution was added with chloroform, stirred vigorously, and then centrifuged at ×8,000g / 20°C / 5 minutes to recover the supernatant. The recovered supernatant was passed through a 0.2μm filter, and the filtrate was used as a phage purification solution.

[0807] As a phage dispersion containing one type of phage, a phage having a titer of about 10 9 The purified phage solution was diluted with sterile tap water at a concentration of about PFU / mL.

[0808] As a phage dispersion liquid containing a plurality of phages, the following dispersion liquid was used: a dispersion liquid containing a plurality of phages at a titer of about 10 9 The liquid obtained by diluting the purified liquids of the phages adjusted to the same level of titer with sterile tap water in equal amounts at about PFU / mL. The phage having the genomic DNA sequence of sequence number 8 as the first phage, the phage having the genomic DNA sequence of sequence number 13 as the second phage, the phage having the genomic DNA sequence of sequence number 14 as the third phage, the phage having the genomic DNA sequence of sequence number 17 as the fourth phage, the phage having the genomic DNA sequence of sequence number 18 as the fifth phage, the phage having the genomic DNA sequence of sequence number 23 as the sixth phage, the phage having the genomic DNA sequence of sequence number 28 as the seventh phage, and the phage having the genomic DNA sequence of sequence number 32 as the eighth phage were used.

[0809] (2) Preparation of bacterial dispersion solution

[0810] In this experiment, the bacterial culture solution prepared in (1) was used to prepare the bacterial dispersion solution used for the treatment of plant infection. The bacterial culture solution diluted to about 10,000 times with sterile tap water was applied to a YPG Agar plate and incubated in an incubator set at 25°C for about 1 to 3 days. The colonies on the YPG Agar plate were suspended with sterile tap water, and the recovered bacterial suspension was measured at the final OD 600 Dilute with sterile tap water to about 0.5 to prepare a bacterial dispersion solution.

[0811] (3) Plant samples

[0812] Commercially available peach saplings (variety: Kawanakajima, one-year-old) were grown in a greenhouse, and saplings grown to a state of having about 100 leaves were used as evaluation samples.

[0813] (4) Phage administration and infection management

[0814] In the phage administration group, for each sample, the phage spray solution was sprayed on the leaves twice with an interval of 2 to 3 days before and after the bacterial infection treatment, and then the bacterial spray solution was sprayed on the leaves 2 to 3 days later, thereby infecting the sample with bacteria. The infected sample was placed in a plastic greenhouse with high humidity for about 2 days after the infection treatment. Then, the phage spray solution was sprayed on the leaves twice again with an interval of 2 to 3 days. In the untreated group, only sterilized tap water was used instead of the phage purified solution, and the treatment was carried out according to the same steps as above.

[0815] (5) Determination of morbidity

[0816] When a certain degree of disease was observed in the untreated group after more than one week from the infection treatment, the disease incidence rate was investigated for the untreated group and each phage-distributed group.

[0817] Leaves with brown spots on the leaf surface, which are characteristic of peach bacterial puncture, were judged as diseased leaves, and the ratio of the number of diseased leaves to the total number of leaves was calculated as the incidence rate. The relative incidence rate of the phage-administered group was calculated as a relative value when the incidence rate of the untreated group was set to 100%.

[0818] (result)

[0819] The results are shown in Fig.21 and 22. The incidence rate of the untreated group was about 36%. On the other hand, the relative incidence rate, with the incidence rate of the untreated group set to 100%, was about 50% on average when a phage dispersion containing one type of phage was applied. When phages were applied, the relative incidence rate was as high as about 61% (the group applied with the 6th phage and the group applied with the 8th phage). On the other hand, the relative incidence rate was the lowest at about 24% in the group applied with the 7th phage.

[0820] In addition, the incidence rate was further reduced when the phage dispersion containing two or more phages was applied compared to the case where the phage dispersion containing one phage was applied. The incidence rate of the untreated group was about 15%. When the phages were applied in combination, the relative incidence rate was the highest at about 41% (a combination of the second phage, the fourth phage, the sixth phage, and the eighth phage). On the other hand, when the first phage to the second phage were combined, the relative incidence rate was the lowest at about 34%.

[0821] From the above, it can be seen that when actually applied to plants, the bacteriophage of the present invention can effectively prevent and control plants from disease infection. In addition, it can be seen that by combined application, a higher effect can be obtained. In addition, it can be seen that the disease control effect of the bacteriophage of the present invention is also effective under the condition that the disease incidence rate in the untreated group is low.

[0822] In chemical pesticides including common antibiotics, there are examples of leaf damage such as yellowing of leaf tips. However, in the group treated with phages, such leaf damage, which is considered to be a significant adverse effect on plants caused by phages, was not observed. This shows that the application of the phages of the present invention has few side effects and is an effective disease control method.

[0823] <Example 16: Disease control effect test of cauliflower black rot>

[0824] (Purpose)

[0825] New phages isolated and having lytic activity against pathogenic bacteria of plant diseases are verified for their effect on plant diseases when applied to plants.

[0826] (method)

[0827] (1) Preparation of phage dissemination fluid

[0828] The bacterium used as the bacterial cell was Xanthomonas campestris pv. campestris (MAFF No. 106765), and the phage dispersion liquid was prepared according to the description of Example 15 except that the following phage was used.

[0829] In this example, a phage having a genomic DNA sequence of sequence number 7 was used as the first phage ( Fig.23 , Φ1), a phage having a genomic DNA sequence of sequence number 13 as a second phage ( Fig.23 , Φ2-1) and a bacteriophage having a genomic DNA sequence of sequence number 47 ( Fig.23 2-2), a phage having a genomic DNA sequence of sequence number 28 as the seventh phage ( Fig.23 7-1) and a bacteriophage having a genomic DNA sequence of sequence number 53 ( Fig.23 7-2), a phage having a genomic DNA sequence of sequence number 41 as the 9th phage ( Fig.23 9), a phage having a genomic DNA sequence of sequence number 44 as the 10th phage ( Fig.23 Middle, Φ10).

[0830] (2) Preparation of bacterial dispersion solution

[0831] A phage dispersion liquid was prepared according to the description of Example 15 except that the bacterial cells were the above-mentioned bacteria.

[0832] (3) Plant samples

[0833] Commercially available cauliflower seeds were sown and grown in a greenhouse, and seedlings with five or more leaves were used as evaluation samples.

[0834] (4) Phage administration and infection management

[0835] Proceed as described in Example 15.

[0836] (5) Determination of morbidity

[0837] The procedure described in Example 15 was followed except that the evaluation was performed 16 days after infection treatment.

[0838] (result)

[0839] The results are shown in Fig.23 The morbidity rate of the untreated group was about 52%. On the other hand, the relative morbidity rate, with the morbidity rate of the untreated group set as 100%, was about 62% on average when a phage dispersion containing one phage was applied. The relative morbidity rate was as high as about 66% when a phage having a genomic DNA sequence of sequence number 28, which was the seventh phage, was applied. Fig.23 On the other hand, in the group in which the phage having the genomic DNA sequence of sequence number 47 was administered as the second phage ( Fig.23The relative incidence rate was the lowest in the group with medium and Φ2-2, which was about 56%.

[0840] From these results, it was confirmed that the 9th and 10th phages exhibited lytic activity even though the specific bacterial strains were different from the strains belonging to Xanthomonas campestris pv. Campestris in which the lytic activity was confirmed in Examples 9 and 10, respectively.

[0841] In addition, when a phage dispersion containing two or more phages was used, the relative incidence rate was further reduced to about 53% on average. The highest relative incidence rate was observed when the 9th phage ( Fig.23 9) and the 10th phage ( Fig.23 On the other hand, when the first phage ( Fig.23 , Φ1), 2 second phages ( Fig.23 , Φ2-1 and Φ2-2) and the 10th phage ( Fig.23 In the case of a total of four phage combinations (medium, Φ10), the relative incidence rate was the lowest, at about 47%.

[0842] From the above, it can be seen that when actually applied to plants, the bacteriophage of the present invention can effectively prevent and control plants from disease infection regardless of the type of plant itself. In addition, it can be seen that by combining the bacteriophage of the present invention, a higher effect can be obtained.

[0843] In the phage-administered group, no significant adverse effects on plants, such as phytotoxicity on leaves, were observed, which could be considered to be caused by the phage. This shows that the administration of the phage of the present invention has few side effects and is an effective disease control method.

[0844] <Example 17: Disease control effect test of tomato bacterial spot disease>

[0845] (Purpose)

[0846] New phages isolated and having lytic activity against pathogenic bacteria of plant diseases are verified for their effect on plant diseases when applied to plants.

[0847] (method)

[0848] (1) Preparation of phage dissemination fluid

[0849] The bacterium used as the bacterial cell was Xanthomonas campestris pv. vesicatoria (MAFF No. 301256), and the phage dispersion liquid was prepared according to the description of Example 15 except that the following phage was used.

[0850] In this example, a phage having a genomic DNA sequence of sequence number 7 was used as the first phage ( Fig.24 , Φ1), a phage having a genomic DNA sequence of sequence number 13 as a second phage ( Fig.24 , Φ2-1), a bacteriophage having a genomic DNA sequence of sequence number 47 ( Fig.24 , Φ2-2) and a bacteriophage having a genomic DNA sequence of sequence number 48 ( Fig.24 2-3), a phage having a genomic DNA sequence of sequence number 28 as the seventh phage ( Fig.24 7), a phage having a genomic DNA sequence of sequence number 44 as the 10th phage ( Fig.24 Middle, Φ10).

[0851] (2) Preparation of bacterial dispersion solution

[0852] A phage dispersion liquid was prepared according to the description of Example 15 except that the bacterial cells were the above-mentioned bacteria.

[0853] (3) Plant samples

[0854] Commercially available tomato seeds were sown and grown in a greenhouse, and seedlings with 50 or more leaves were used as evaluation samples.

[0855] (4) Phage administration and infection management

[0856] Proceed as described in Example 15.

[0857] (5) Determination of morbidity

[0858] The ...

Claims

1. A bacteriolytic agent comprising a bacteriophage comprising, in its genomic DNA, a gene comprising an amino acid sequence selected from any one of SEQ ID NOs: 42, 11, 45, and 15 and encoding a tail fiber protein having target bacterium recognition activity.

2. The bacteriolytic agent according to claim 1, wherein The gene comprises a base sequence selected from any one of SEQ ID NOs: 43, 12, 46, and 16.

3. The bacteriolytic agent according to claim 1, wherein The base sequence of the genomic DNA includes a base sequence selected from any one of SEQ ID NOs: 44, 13, 47, 48, and 17.

4. A bacteriolytic agent comprising a bacteriophage, wherein the bacteriophage comprises in its genomic DNA a gene comprising the amino acid sequence shown in SEQ ID NO: 1 and encoding a tail fiber protein having target bacterium recognition activity.

5. The bacteriolytic agent according to claim 4, wherein The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence represented by any one of SEQ ID NOs: 2 to 4. The bacteriolytic agent according to claim 4, wherein The gene includes the base sequence shown in any one of SEQ ID NOs: 5 to 7.

7. The bacteriolytic agent according to claim 4, wherein The base sequence of the genomic DNA includes the base sequence represented by any one of SEQ ID NOs: 8 to 10.

8. A bacteriolytic agent comprising a bacteriophage having a genomic DNA sequence comprising a base sequence selected from the group consisting of SEQ ID NOs: 14, 18, 19, and 32 to 36.

9. A bacteriolytic agent comprising a bacteriophage comprising a gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 21 in its genomic DNA.

10. The bacteriolytic agent according to claim 9, wherein The gene comprises the base sequence shown in SEQ ID NO:

22. The bacteriolytic agent according to claim 9, wherein The base sequence of the genomic DNA includes the base sequence shown in sequence number 23.

12. A bacteriolytic agent comprising a bacteriophage, said bacteriophage comprising in its genomic DNA: A gene comprising the amino acid sequence shown in SEQ ID NO: 24, 49, or 37 and encoding a tail tube protein A having target bacteria recognition activity; and A gene comprising an amino acid sequence selected from any one of SEQ ID NOs: 57, 50, 38, 54, and 59, and encoding a tail tube protein B having target bacteria recognition activity.

13. The bacteriolytic agent according to claim 12, wherein The gene encoding the tail tube protein A comprises the base sequence shown in SEQ ID NO: 26, 51 or 39. The bacteriolytic agent according to claim 12 , wherein The gene encoding the tail pipe protein B comprises a base sequence selected from any one of SEQ ID NOs: 27, 52, 40, and 55.

15. The bacteriolytic agent according to claim 12, wherein The base sequence of the genomic DNA includes a base sequence selected from any one of SEQ ID NOs: 28, 29, 53, and 41. The bacteriolytic agent according to any one of claims 1 to 15, which exhibits bacteriolytic activity against bacteria of the genus Xanthomonas. A composition comprising the bacteriolytic agent according to claim 16 as an active ingredient.

18. A plant disease control composition comprising the composition according to claim 17.

19. The plant disease control composition according to claim 18, which is a plant disease control composition for plant diseases caused by bacteria belonging to the genus Xanthomonas.

20. The plant disease control composition according to claim 18, comprising other bacteriophages showing bacteriolytic activity against bacteria of the genus Xanthomonas.

21. A method for preventing and controlling plant diseases, comprising: The contacting step comprises contacting the plant disease control composition according to claim 18 with the target plant.

22. A method for identifying bacteria of the genus Xanthomonas, the method comprising: a culturing step of culturing the test bacteria separated from plant tissues infected by plant diseases to obtain a culture; a mixing step of mixing the culture with the bacteriolytic agent according to any one of claims 1 to 16 to obtain a mixture; a mixture cultivation step of cultivating the mixture under given conditions; and The determination step comprises determining that the test bacteria are Xanthomonas bacteria when the test bacteria undergo lysis after the mixture culturing step.

23. The method according to claim 22, wherein In the mixture culturing step, the mixture further comprises a liquid culture medium containing soft agar, and the mixture is cultured on a solid culture medium.

24. The method according to claim 22, wherein In the culturing step, the culture comprises a liquid medium containing soft agar, and the culture is cultured on a solid medium.

25. The method according to any one of claims 22 to 24, further comprising, before the culturing step: The isolation step is to separate the test bacteria from plant tissues infected by plant diseases.

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