Bacteriophage, bacterial wilt disease control agent, and bacterial wilt disease control method

[0001] The present application is the national stage of international application PCT/JP2020/029189, filed on Jul. 30, 2020.

[0002] The present invention relates to a bacteriophage, a bacterial wilt disease control agent and a bacterial wilt disease control method.

[0003] Ralstonia solanacearum infects 200 plant species or more such as the Solanaceae plants and causes bacterial wilt. When the disease progresses, the plant infected with Ralstonia solanacearum withers.

[0004] Major chemical pesticides which have been used so far against bacterial wilt are chloropicrin and bromomethane, which are powerful drugs.

[0005] In view of the problems such as the increase in the effective application amounts, the environmental pollution, the ozone layer depletion, the influences on the health and the pesticide residues, however, development of safe alternative pesticides and control techniques which replace the chemical pesticides is desired.

[0006] Although techniques using bacteriophages (PTLs 1 to 3) have been known so far as safe alternative pesticides and control techniques which replace the chemical pesticides for example, the techniques are effective against a limited number of Ralstonia solanacearum strains, and a technique which is effective against a wider variety of Ralstonia solanacearum strains has been desired.

• • [0007] PTL 1: JP-A-2007-252351
  • [0008] PTL 2: JP-A-2018-24589
  • [0009] PTL 3: WO2017/104347

[0010] Accordingly, a problem of the invention is to provide a bacteriophage which is effective against a wider variety of Ralstonia solanacearum strains and a bacterial wilt disease control method using the bacteriophage.

[0011] As a result of extensive studies to solve the problem, the present inventors have found that a bacteriophage in which the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site is a specific sequence infects a wide variety of Ralstonia solanacearum and thus have completed the invention.

[0012] That is, the invention is a bacteriophage characterized in that the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site is the amino acid sequence of SEQ ID NO: 1 and that the bacteriophage infects Ralstonia solanacearum.

[0013] Moreover, the invention is a bacterial wilt disease control agent characterized by containing the bacteriophage as an active component.

[0014] Furthermore, the invention is a bacterial wilt disease control method in a plant characterized by administering the bacterial wilt disease control agent to a plant or a plant growth medium.

[0015] Further, the invention is the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site shown in SEQ ID NO: 1 and a base sequence encoding the amino acid sequence.

[0016] The bacteriophage of the invention infects a wide variety of Ralstonia solanacearum strains and thus is effective for controlling bacterial wilt.

[0017] Moreover, by incorporating (or substituting with) the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site of the invention (SEQ ID NO: 1) into a host recognition site of a tail fiber protein of another bacteriophage or the like by a general method using the amino acid sequence or a base sequence encoding the amino acid sequence, infection of a wide variety of Ralstonia solanacearum strains is enabled.

[0026] The amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site of the bacteriophage of the invention is the amino acid sequence of SEQ ID NO: 1, and the bacteriophage infects Ralstonia solanacearum.

[0027] The amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site of the bacteriophage shown in SEQ ID NO: 1 is largely different from the amino acid sequences at the C-terminus of a tail fiber protein including a host recognition site of conventionally known bacteriophages, and as a result, the bacteriophage infects a wide variety of Ralstonia solanacearum strains. For example, the identities of the amino acid sequences at the C-terminus of a tail fiber protein including a host recognition site of RSJ5 and RSB2, which are related bacteriophages of the bacteriophage of the invention, to the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site of the bacteriophage of the invention are only 38% and 23% according to ClustalW analysis (see the Examples).

[0028] As described above, the bacteriophage of the invention infects a wide variety of Ralstonia solanacearum strains because the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site is the amino acid sequence of SEQ ID NO: 1.

[0029] Here, by incorporating (or substituting with) the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site of the invention (SEQ ID NO: 1) at a host recognition site of a tail fiber protein of another bacteriophage or the like by a general method using the amino acid sequence or a base sequence encoding the amino acid sequence, infection of a wide variety of Ralstonia solanacearum strains is enabled (Document 1. (Ando H, Lemire S, Pires D P, Lu T K. 2015. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Systems 1:187-196.)). The base sequence encoding the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site (SEQ ID NO: 1) which the bacteriophage of the invention has is not particularly limited as long as the base sequence corresponds to the amino acid sequence but is preferably the sequence from the 34, 313th to the 35, 968th residues of the base sequence of SEQ ID NO: 3 (the whole genome sequence of RKP181)

[0030] The Ralstonia solanacearum infected with the bacteriophage of the invention is not particularly limited as long as the Ralstonia solanacearum is any of those found from 200 plant species or more such as the Solanaceae plants, the Zingiberaceae plants and the plants, the Lamiaceae but bacteriophage infects at least all of strain MAFF107624, strain MAFF211266, strain MAFF211270, strain MAFF211543, strain MAFF301859, strain MAFF311644, strain MAFF730103, strain MAFF730131, strain MAFF302745, strain MAFF311632, strain MAFF211536, strain MAFF331041, strain MAFF730139, strain MAFF211280, strain MAFF211533, strain MAFF211468, strain MAFF211516, strain MAFF311101, strain MAFF311102, strain MAFF211479, strain MAFF211471, strain MAFF211483, strain MAFF211484, strain MAFF211486, strain MAFF211272, strain MAFF211276, strain MAFF211278, strain MAFF211490, strain MAFF211492, strain MAFF211497, strain MAFF211476, strain MAFF211414, MAFF211429 strain strain and MAFF301558 and further infects the 70 Ralstonia solanacearum strains or more collected by the present inventors. In this regard, the Ralstonia solanacearum strains having a number with MAFF can be obtained from Genebank Project, NARO, Agriculture National and Food Research Organization.

[0031] In this regard, the infection here refers to the state in which plaques are formed on an agar plate on which Ralstonia solanacearum grows or the state of lysing in a liquid medium in the presence of RKP181.

[0032] The bacteriophage of the invention described above can be obtained by screening known bacteriophages according to a general method to find those in which the amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site is the amino acid sequence of SEQ ID NO: 1.

[0033] Moreover, the bacteriophage of the invention should have the above amino acid sequence. Thus, the bacteriophage of the invention may belong to any group, any order or any family but preferably belongs to the family Podoviridae of the order Caudovirales of the group I.

[0034] The bacteriophage of the invention has a head diameter of 30 to 90 nm, preferably 55 to 66 nm, a tail length of 5 to 30 nm, preferably 11 to 17 nm and a width of 5 to 20 nm, preferably 10 to 17 nm.

[0035] The bacteriophage of the invention has a double-stranded genome.

[0036] The genome size of the bacteriophage of the invention is not particularly limited but is for example 6,000 to 280,000 bp, preferably 38,000 to 40,000 bp. The genome size is the value of the whole genome base sequence.

[0037] The GC content of the bacteriophage of the invention is not particularly limited but is for example 55 to 75%, preferably 61 to 64%. The GC content is the value calculated from the whole genome base sequence.

[0038] The number of genes in the bacteriophage of the invention is not particularly limited but is for example 10 to 330 genes, preferably 50 genes. Of the 50 genes, 49 genes are in the same direction.

[0039] The genomic DNA of the bacteriophage of the invention is preferably digested into fragments through treatment with the restriction enzyme Eco81I, particularly preferably into five fragments of 2 kb or more. The conditions for treatment with the restriction enzyme Eco81I are at 35 to 40° C. for one to three hours.

[0040] Furthermore, the bacteriophage of the invention may have, for example, genes such as lysozyme and typeII holin or may have a circular or linear genome.

[0041] A preferable example of the bacteriophage of the invention explained above is RKP181 found by the present inventors. RKP181 was deposited for an international deposit at NITE Patent Microorganisms Depositary, National Institute of Technology and Evaluation (address: #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) on Mar. 27, 2020 under NITE BP-03186.

[0042] The bacteriophage of the invention also includes those having modification, mutation or the like while maintaining the properties.

[0043] Because the bacteriophage of the invention can infect a wide variety of Ralstonia solanacearum, a bacterial wilt disease control agent can be obtained using the bacteriophage as an active component.

[0044] In this regard, the control here refers to suppression and alleviation of infection of a plant with Ralstonia solanacearum, suppression and alleviation of the onset of bacterial wilt by Ralstonia solanacearum, suppression and alleviation of spread of infection with bacterial wilt disease or control of Ralstonia solanacearum.

[0045] The amount of the bacteriophage of the invention in the bacterial wilt disease control agent may be appropriately set depending on the purpose of control, but for example, the bacteriophage of the invention may be contained at potency of 10³ to 10¹² pfu/mb, preferably 10⁵ to 10¹¹ pfu/mL.

[0046] The bacterial wilt disease control agent may contain only the bacteriophage of the invention, which is the active component, but may further contain another substance, a composition of the like such as an agronomically, pharmacologically and botanically acceptable protein stabilizer.

[0047] The bacterial wilt disease control agent described above can control bacterial wilt disease in a plant when administered to a plant or a plant growth medium. Examples of the plant include tomatoes, eggplants, bell peppers and tobaccos of the family Solanaceae, Perilla frutescens var. crispa and Perilla frutescens of the family Lamiaceae, ginger, turmeric and curcuma of the family Zingiberaceae and the like. The plant growth medium may be soil, a solid medium such as a mat containing organic materials, a liquid containing a nutrient solution or the like. The method and the conditions for administering the bacterial wilt disease control agent to such a plant or a plant growth medium are not particularly limited but are, for example, spraying or dropping to the plant or the plant growth medium, injection into the plant or the like.

[0048] The invention is explained in detail below referring to Examples, but the invention is not limited to the Examples.

Separation of Bacteriophage:

[0049] Soil was suspended in water and then left still, and thus a supernatant was obtained. The supernatant was filtered with a filter of 0.25 μm, and the filtrate was subjected to a plaque assay using Ralstonia solanacearum as the host. By isolating a formed plaque, bacteriophage RKP181 was obtained.

Analysis of Bacteriophage RKP181:

(1) Electron Microscope Analysis

[0050] A phage suspension of 2×10¹¹ pfu/ml was stained with 1% uranyl acetate, and pictures were taken under an electron (JEM-1400Pus, manufactured by JEOL Ltd.).

(2) Determination of Genome Size

[0051] The genomic DNA was prepared from the phage particles by phenol/chloroform extraction. To determine the size of the genome, the purified genomic DNA was subjected to agarose gel electrophoresis with Mupid-2plus electrophoresis apparatus (manufactured by Mupid) using 0.3% agarose (Agarose H, manufactured by Nippon Gene Co., Ltd.). Moreover, the genomic DNA which was treated with restriction enzymes EcoRI the (manufactured by Toyobo Co., Ltd.) and Eco81I (manufactured by Takara Bio Inc.), respectively was subjected to agarose gel electrophoresis with Mupid-2plus electrophoresis apparatus using 0.8% agarose (Agarose 1200 Standard Type, manufactured by PH Japan Co., Ltd) or 0.3% agarose (Agarose H, manufactured by Nippon Gene Co., Ltd.).

[0052] According to the bands of the size marker, the genome size of RKP181 was estimated to be 38,400 to 48,500 bp. Moreover, as a result of comparison with the bands of the size marker, the band sizes obtained by the treatment with EcoRI were 21.1 kb, 16.1 kb, 1.3 kb and 1 kb, and the band sizes obtained by the treatment with Eco81I were 12.9 kb, 10.2 kb, 7 kb, 6.3 kb and 2 kb, which means that there were five fragments of 2 kb or more. It was thus found that the genome size of RKP181 is 38.4 to 39.5 kbp.

(3) Determination of Genomic Structure

[0053] The purified genomic DNA was treated with a single-stranded DNA nuclease (S1 Nuclease, manufactured by Promega Corporation), a DNA nuclease (Recombinant DNase I, manufactured by Takara Bio Inc.), an RNA nuclease (RNase A, manufactured by Takara Bio Inc.) and a linear DNA nuclease (BAL31 nuclease, manufactured by New England Biolabs, Inc.) and subjected to electrophoresis by the method described in the above Example. Moreover, the terminal structures of the genome were determined by treating the genomic DNA which was treated with the linear DNA nuclease (BAL31 nuclease) for different time periods with Eco81I (Takara Bio Inc.) and conducting electrophoresis by the method described in the above Example.

[0054] Because the genomic DNA of RKP181 was not digested with the single-stranded DNA nuclease (S1 Nuclease) and the RNA nuclease (RNase A) but was digested with the DNA nuclease (Recombinant DNase I) and the linear DNA nuclease (BAL31 nuclease), it was found that the genome of RKP181 is linear double-stranded DNA. Moreover, the fragments of 12.9 kb, 10.2 kb, 7 kb, 6.3 kb and 2 kb obtained by the treatment with Eco81I were lost with time with the linear DNA nuclease (BAL31 nuclease) from the fragments of 10.2 kb and 6.3 kb,

(4) Genomic Analysis

[0055] The whole genome base sequence of RKP181 was determined with PacBio RS II (manufactured by PACIFIC BIOSCIENCES), The determined base sequences were assembled using RS_HGAP Assembly 2.3.0. To estimate the terminal structures and the lengths of the terminal sequences of the genome, PhageTerm (Galaxy) was used. The terminal redundant sequences were determined by Sanger sequencing (Applied Biosystems 3730xl DNA analyzer). Related strains based on the whole genome sequences were searched by homology search with BLASTN (Table 1). The open reading frames (ORFs) were estimated using MetaGeneAnnotator 1.0 of MiGAP ver2.23 (Database Center for Life Science), tRNAscan-SE 1.23 and BLAST 2.2.18 (Table 2). The functions of the detected ORFs were estimated using the COG, Refseq, TrEMBL and nr databases for those at the top obtained by homology search with MiGAP/BLAST and PSI-BLAST (Table 2). The molecular phylogenetic tree based on the amino acid sequences of the DNA packaging protein B gene was created by the maximum likelihood method by obtaining the amino acid sequences of the DNA packaging protein B gene of the related strains from the NCBI database and aligning the sequences with ClustalW of MEGA 7.0.26 (FIG. 2).

[0056] The genome had a size of 39, 455 bp and contained redundant sequences at both ends, and the terminal redundant sequences were 239 bp in length. The GC content of the genome was 62.6%. From the identity comparison of the genomes, RKP181 was related to phages of the genus 7-like virus of the family Podoviridae (podovirus) of the order Caudovirales, which is consistent with the results of the electron microscope analysis (Table 1 and FIGS. 1 and 2). The closest related strain was known Ralstonia phage strain phiITL-1 (Table 1 and FIG. 2), and the identity was 87% (coverage of 84%) according to BLASTN. The next homologous strain was Ralstonia phage strain RSB2 (coverage of 70% and identity of 77%). From the results of the analysis with PhageTerm and the phylogenetic systematic analysis based on the DNA packaging DNA protein B gene (FIG. 2), the terminal structures of the genome of RKP181 were estimated to have a short direct terminal redundancy [short DTR] structure and to be T7 type as described above. The whole genome of RKP181 determined with PacBio RS II is shown in SEQ ID NO: 3. Moreover, as shown in Table 2, the genome of RKP181 included 50 genes in total, of which 49 genes were in the same direction.

[0057]

TABLE 1
								Base	GC	Gene
Top	Accession	Species	Coverage	Identity	Family	Genus	Structure	Length (b)	(%)	Number
1	KP343639.1	Ralstonia	84%	87%	Podoviridae	T7 virus	Circular	38,731	62.8%	54
		phage philTL-1
2	AB597179.1	Ralstonia	70%	77%	Podoviridae	T7 virus	Linear	40,411	61.8%	53
		phage RSB2
3	KR153873.1	Delftia	6%	75%	Podoviridae	T7 virus	Circular	38,084	60.4%	48
		phage IME-DE1
4	MF893341.1	Ralstonia	3%	74%	Podoviridae	T7 virus	Circular	39628	61.6%	43
		phage RPSC1

[0058]

TABLE 2
											homolog
	Start	End	Length	Length							accession
ORF	(nt)	(nt)	(nt)	(aa)	strand	putative function	organism	product	E-value	Ident.	no.
DTR	1	239	239	NA	+	direct terminal	NA	NA	NA	NA	NA
						repeat
1	404	634	231	76	−	Signal transduction	Ralstonia	hypothetical	3E−39	84%	AJT60839.1
						histidine kinase	phage	protein
							philTL-1
2	1,304	1,504	201	66	+	NA	Ralstonia	hypothetical	4E−30	76%	AJT60799.1
							phage	protein
							philTL-1
3	1,769	2,011	243	80	+	NA	Ralstonia	hypothetical	2E−34	70%	AJT60800.1
							phage	protein
							philTL-1
4	2,022	2,441	420	139	+	Cell division	Ralstonia	hypothetical	5E−58	63%	YP_009017724.1
						protein Ftsl/	phage	protein ORF3
						penicillin-	RSB2
						binding protein 2
5	2,519	2,764	246	81	+	Non-ribosomal	Ralstonia	hypothetical	5E−35	70%	YP_009017725.1
						peptide synthetase	phage	protein ORF4
						modules and	RSB2
						related proteins
6	2,820	3,002	183	60	+	NA	Ralstonia	hypothetical	1E−14	52%	YP_009017726.1
							phage	protein ORF5
							RSB2
7	2,999	3,178	180	59	+	NA	Ralstonia	hypothetical	8E−16	65%	YP_009017727.1
							phage	protein ORF6
							RSB2
8	3,175	3,495	321	106	+	Uncharacterized	Ralstonia	hypothetical	2E−26	59%	YP_009017728.1
						bacitracin	phage	protein ORF7
						resistance protein	RSB2
9	3,492	3,857	366	121	+	Phosphoenol-	Ralstonia	hypothetical	1E−49	72%	YP_009017729.1
						pyruvate	phage	protein ORF8
						carboxykinase	RSB2
						(GTP)
10	3,870	4,049	180	59	+	NA	Ralstonia	hypothetical	6E−06	48%	AJT60804.1
							phage	protein
							philTL-1
11	4,075	4,341	267	88	+	NA	Ralstonia	hypothetical	1E−37	73%	AJT60805.1
							phage	protein
							philTL-1
12	4,320	4,586	267	88	+	Ribonucleases	Ralstonia	hypothetical	1E−39	78%	AJT60806.1
						G and E	phage	protein
							philTL-1
13	4,651	5,007	357	118	+	NA	Ralstonia	hypothetical	4E−46	85%	AJT60808.1
							phage	protein
							philTL-1
14	5,058	5,466	408	135	+	Enoyl reductase	Ralstonia	hypothetical	1E−42	77%	AJT60810.1
						domain of yeast-	phage	protein
						type FAS1	philTL-1
15	5,489	5,570	102	33	+	NA	Ralstonia	hypothetical	1E−12	91%	YP_009017736.1
							phage	protein
							RSB2
16	5,567	6,499	933	310	+	ATP-dependent	Ralstonia	ATP-dependent	7E−110	54%	AJT60812.1
						DNA ligase	phage	DNA ligase
							philTL-1
17	6,496	6,615	120	39	+	NA	Spirosoma	M56 family	0.5	49%	WP_020605153.1
							spitsbergense	metallo-
								peptidase
18	6,768	9,365	2,598	865	+	Mitochondrial	Ralstonia	DNA-directed	0E+00	80%	YP_009017739.1
						DNA-directed RNA	phage	RNA
						polymerase	RSB2	polymerase
19	9,385	9,831	447	148	+	NA	Ralstonia	hypothetical	4E−57	59%	AJT60815.1
							phage	protein
							philTL-1
20	10,012	10,257	246	81	+	NA	Ralstonia	hypothetical	9E−05	53%	AJT60816.1
							phage	protein
							philTL-1
21	10,254	10,553	300	99	+	Flp pilus	Ralstonia	hypothetical	5E−24	62%	AJT60817.1
						assembly	phage	protein
						protein TadC	philTL-1
22	10,616	10,921	308	101	+	NA	Ralstonia	hypothetical	2E−60	89%	AJT60818.1
							phage	protein
							philTL-1
23	10,914	11,609	696	231	+	AraC-type	Ralstonia	hypothetical	6E−93	68%	YP_009017744.1
						DNA-binding	phage	protein
						domain-containing	RSB2	ORF23
						proteins
24	11,590	11,763	174	57	+	deoxynucleoside	Ralstonia	hypothetical	7E−17	67%	AJT60821.1
						monophosphate	phage	protein
						kinase	philTL-1
25	11,831	12,496	666	22	+	NA	Ralstonia	Putative	1E−138	91%	AJT60822.1
							phage	ssDNA binding
							philTL-1	protein
26	12,499	12,966	468	155	+	single-stranded	Ralstonia	T7-like phage	2E−105	94%	AJT60823.1
						DNA-binding	phage	endonuclease
						protein	philTL-1
27	12,968	13,438	471	156	+	Formamido-	Ralstonia	lysozyme	5E−61	63%	YP_009017748.1
						pyrimidine-	phage	[Peplidoglycan
						DNA glycosylase	RSB2	recognition
								proteins]
28	13,545	15,230	1,586	561	+	lysozyme(N-	Ralstonia	DNA primase/	0E+00	90%	AJT60826.1
						acetylmuramoyl-L-	phage	helicase-like
						alanine amidase)	philTL-1	protein
29	15,240	17,348	2,109	702	+	Replicative	Ralstonia	DNA	0E+00	95%	AJT60827.1
						DNA helicase	phage	polymerase″
							philTL-1
30	17,398	17,775	378	125	+	DNA polymerase	Ralstonia	hypothetical	8E−56	78%	AJT60828.1
						I - 3′-5′	phage	protein
						exonuclease and	philTL-1
						polymerase
						domains
31	17,798	17,998	201	56	+	NA	Pectobacterium	HNS binding	2E−18	55%	ATN92966.1
							phage	protein
							PPWS4
32	17,995	18,879	885	294	+	Glycosyltransferase	Ralstonia	phage	0E+00	95%	AJT60830.1
							phage	exonuclease
							philTL-1
33	18,901	19,212	312	103	+	5′-3′ exonuclease	Ralstonia	hypothetical	7E−36	67%	AJT60831.1
						(including	phage	protein
						N-terminal	philTL-1
						domain of Poll)
34	19,226	19,591	366	121	+	Fe2+ transport	Ralstonia	hypothetical	3E−63	76%	AJT60832.1
						system protein B	phage	protein
							philTL-1
35	19,594	19,896	303	100	+	Uncharacterized	Ralstonia	hypothetical	2E−27	82%	AJT60833.1
						conserved protein	phage	protein
							philTL-1
36	19,901	21,496	1,596	531	+	Small-conductance	Ralstonia	Putative head	0E+00	92%	AJT60834.1
						mechanosensitive	phage	to tail joining
						channel	philTL-1	protein (collar)
37	21,557	22,381	825	274	+	Transcriptional	Ralstonia	Putative	1E+00	84%	AJT60835.1
						regulators	phage	capsid
							philTL-1	assembly
								protein
38	22,494	23,453	960	319	+	Phage T7 capsid	Ralstonia	Putative	0E+00	93%	AJT60836.1
						assembly protein	phage	major capsid
							philTL-1	protein
39	23,560	24,138	579	192	+	major capsid	Ralstonia	Putative tail	8E−123	87%	AJT60837.1
						protein	phage	tubular
							philTL-1	protein A
40	24,141	25,507	2,367	788	+	T7 tail tubular	Ralstonia	Putative tail	0E+00	80%	YP_009017762.1
						gp11 protein	phage	tubular
							RSB2	protein B
41	26,615	27,079	465	154	+	FOG: WD40 repeat	Ralstonia	Putative	2E−104	94%	AJT60786.1
							phage	internal
							philTL-1	virion
								protein A
42	27,072	27,653	582	193	+	Putative internal	Ralstonia	Putative	2E−108	87%	AJT60787.1
						virion protein A	phage	internal
							philTL-1	virion
								protein B
43	27,663	29,846	2,184	727	+	Putative internal	Ralstonia	Putative	0E+00	83%	AJT60788.1
						virion protein B	phage	internal
							philTL-1	virion
								protein C
44	29,865	33,749	3,885	1,294	+	Putative internal	Ralstonia	Putative	0E+00	88%	AJT60789.1
						virion protein C	phage	internal
							philTL-1	virion
								protein D
45	33,833	35,958	2,136	711	+	Soluble lytic	Ralstonia	putative	4E−107	42%	YP_009218128.1
						murein	phage RSJ5	phage
						transglycosylase		tail fiber
						and related		protein
						regulatory proteins
						(some contain
						LysM/invasin
						domains)
46	35,968	36,294	327	108	+	Phage T7 tail	Ralstonia	Putative	4E−68	96%	AJT60793.1
						fibre protein	phage	uncharacterized
							philTL-1	protein
47	36,294	36,506	213	70	+	AraC-type DNA-	Ralstonia	Putative	4E−35	84%	AJT60794.1
						binding domain-	phage	lysis protein
						containing proteins	philTL-1
48	36,503	36,775	273	90	+	type II holin	Ralstonia	Putative	4E−35	84%	AJT60794.1
							phage	packaging
							philTL-1	maturation
								protein A
49	36,798	38,579	1,782	593	+	DNA packaging	Ralstonia	Putative	0E+00	94%	AJT60797.1
						protein, small	phage	packaging
						subunit	philTL-1	maturation
								protein B
50	38,795	39,007	213	70	+	NA	Ralstonia	hypothetical	5E−20	77%	AJT60798.1
							phage	protein
							philTL-1
DTR	39,217	39,455	239	NA	+	Direct terminal	NA	NA	NA	NA	NA
						repeat

(5) Genetic Map

[0059] Maps of major genes in the genomes were drawn using PHASTER, and RKP181 and Ralstonia phage RSB2 (AB597179.1) of JP-A-2018-24589 were compared (FIG. 3). Moreover, the entire base sequences were also compared using comparative genomic software LAGAN (FIG. 4).

[0060] As in RSB2, the genome of RKP181 included three distinct function modules of class I, class II and class III (FIG. 3, Document 2 (Kawasaki T, Narulita E, Matsunami M, Ishikawa H, Shimizu M, Fujie M, Bhunchoth A, Phironrit N, Chatchawankanphanich O, Yamada T. 2016. Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum. 492:73-81.) Virology and Document 3 (JP-A-2018-24589)). Moreover, because the T-type RNA polymerase gene (RNAP) is in class I in the genome of RKP181 as in RSB2, it is believed that the infection cycle period is shorter than those of other bacteriophages due to rapid expression of RNA polymerase like RSB2 (FIG. 3, see below).

[0061] Although the whole genome base sequence and the genetic map of RKP181 are similar to those of RSB2, regarding the tail fiber gene, the identity of ORF45 of RKP181 and the homolog of RSB2 is significantly low (see below),

(6) Analysis of Tail Fiber Protein-Encoding Gene (Tail Fiber Gene)

[0062] Homology search of the amino acid sequence of ORF45 (SEQ ID NO: 2) encoded by the tail fiber gene of RKP181 was conducted using BLASTP (Table 3). Moreover, the amino acid sequence of ORF45 and known homologous tail fiber gene products were aligned with ClustalW of MEGA 7.0.26, and a molecular phylogenetic tree was created by the maximum likelihood method (FIG. 5). Further, the domains of ORF45 were searched using BLASTP. The identity comparison of the sequence of the 1-149th amino acids of the phage body (tail tube) linker site of the tail fiber gene (AAM43540.1) of T7 and the 1-148th amino acids of ORF45 (shown in SEQ ID NO: 2) of RKP181 was conducted with BLASTP. Subsequently, homology search of the two identified domains, namely, the sequences of the 1-160th amino acids at the N-terminus and the 161-711th amino acids at the C-terminus, was conducted with BLASTP (Table 4 and Table 5). The identities of the tail fiber gene products of RKP181, T7 and RSB2 were compared by obtaining the sequence (BAJ51834.1) of the tail fiber gene (ORF46) of RSB2 and the sequence (P03748.1) of tail fiber gene gp17 of T7 from the NCBI database and comparing the identities using ClustalW of MEGA 7.0.26 (FIG. 6).

[0063] As a result of the homology search with BLASTP, the identity of ORF45 of RKP181 to the tail fiber protein of Ralstonia phage RSJ5 was the highest (coverage of 64% and identity of 42%) (Table 3). Moreover, the identity to the tail fiber protein of Ralstonia phage RSB2 was 53% (coverage of 53%), and the identity to the tail fiber protein of Ralstonia phage phiITL-1 was 85% (coverage of 22%). The identity to the tail fiber protein of Enterobacteria phage T7 was 42% (coverage of 37%). Furthermore, when the identities of the full-length tail fiber proteins were compared by the analysis with ClustalW, the identity of ORF45 of RKP181 to the tail fiber protein of RSJ5 was 38%, and that to the tail fiber protein of RSB2 was 23%, which were found to be significantly lower than the identities obtained by the base sequence comparison of the whole genomes (Table 1). The analysis results show that the amino acid sequence of ORF45 (shown in SEQ ID NO: 2) encoded by the tail fiber gene of RKP181 is unique (see below).

[0064]

TABLE 3
Top	Protein	Species	Coverage	Identity	Accession
1	putative phage tail fiber protein	Ralstonia phage RSJ5	64%	42%	YP_009218128.1
2	putative tail fiber protein	Ralstonia phage RSB2	53%	53%	YP_009017767.1
3	putative tail fiber protein	Ralstonia phage philTL-1	22%	85%	AJT60790.1
4	tail fibers protein	Escherichia phage 64795_ec1	37%	43%	YP_009291518.1
5	tail fiber protein	Escherichia phage C5	38%	43%	AYD80209.1
6	hypothetical protein	Ralstonia solanacearum	67%	32%	WP_064049197.1
7	tail fiber protein	Escherichia phage HZ2R8	65%	44%	AUV62662.1
8	tail fiber protein	Pectobacterium phage PP74	34%	44%	APD19655.1
9	tail fiber protein	Enterobacteria phage T7	37%	42%	AAM43540.1
10	hypothetical protein	Pseudomonas chlororaphis	20%	64%	WP_053269386.1

[0065] As a result of the comparison of the identities based on the whole genome sequences, RKP181 was related to T7-like bacteriophages, RSB2 and phiITL-1 (Table 1), and similar results were obtained from the molecular phylogenetic analysis based on the amino acid sequences of the DNA packaging protein B gene (FIG. 2). When a molecular phylogenetic tree was created using the amino acid sequences of the tail fiber gene, however, RKP181 belonged to a different clade from that including RSB2, phiITL-1 and T7 unlike in FIG. 2 (FIG. 5).

[0066] The tail fiber gene of bacteriophage T7 has a phage body (tail tube) linker site at the 1-149th amino acids at the N-terminus and has a host recognition site (tip domain) at the 465-553rd amino acids at the C-terminus (Document 4 (Steven A C, Trus B L, Maizel J V, Unser M, Parry D A D, Wall J S, Hainfeld J F, Studier F W, 1988. Molecular substructure of a viral receptor-recognition protein: The gp17 tail-fiber of bacteriophage T7. J Mol Biol 200:351-365.) and Document 5 (Garcia-Doval C, Raaij M J van. 2012. Structure of the receptor-binding carboxy-terminal domain of bacteriophage T7 tail fibers. PNAS 109:9390-9395.)).

[0067] When the domains of ORF45 of RKP181 were searched using BLASTP, the tail fibre protein (pfam03906) of T7 and the chaperone domain (pfam13884) of a phage protein, endosialidase were identified. Regarding the tail fibre protein (pfam03906), an identity of 58% (coverage of 96%) was detected between the sequence of the 1-149th amino acids of the phage body (tail tube) linker site of the tail fiber gene (AAM43540.1) of T7 and the 1-148th amino acids of ORF45 (shown in SEQ ID NO: 2) of RKP181. When BLASTP search was conducted using the sequence of the 1-148th amino acids of ORF45 of RKP181, the identities to the homologous sites of the tail fiber proteins of Ralstonia phage phiITL-1 and Ralstonia phage RSB2 were 85% (coverage of 100%) and 73% (coverage of 100%), respectively. Furthermore, as a result of the analysis using BLASTP and ClustalW, the identities of the 1-160th amino acids of ORF45 of RKP181 were largely different from those of the subsequent sequence at the C-terminus (Tables 4 and 5). In other words, the 1-160th amino acids at the N-terminus of ORF45 of RKP181 had an identity of 85% (coverage of 99%) to the homologous region of the tail fiber protein of phiITL-1, an identity of 72% (coverage of 99%) to that of RSB2 and an identity of 58% (coverage of 89%) to that of T7 (Table 4).

[0068]

TABLE 4
Top	Protein	Species	Coverage	Identity	Accession
1	putative tail fiber protein	Ralstonia phage philTL-1	99%	85%	AJT60790.1
2	putative tail fiber protein	Ralstonia phage RSB2	99%	72%	YP_009017767.1
3	hypothetical protein	Pseudomonas chlororaphis	92%	64%	WP_053269386.1
4	Phage tail fibers	Yersinia phage fPS-59	96%	57%	SOO46827.1
5	putative tail fiber protein	Erwinia phage	90%	59%	QEQ94708.1
		pEp_SNUABM_09
38	tail fiber protein	Escherichia phage T7	89%%	58%	AAM43534.1

[0069] As described above, the N-terminus side of the tail fiber protein, which is the phage body (tail tube) linker site, was widely conserved among known bacteriophages and especially was a domain with a high identity between RKP181 and related bacteriophages. On the other hand, the domain at the C-terminus involving in the host recognition varied among the bacteriophages. The bacteriophages that have a tail fiber gene in which the chaperon domain (pfam13884) of endosialidase is located at the C-terminus and that infect Ralstonia solanacearum, like RKP181, were RSJ5 and RS-PI-1 only. Moreover, when homology search was conducted with BLASTP using the sequence of the 161-711th amino acids of RKP181 excluding the phage body linker site, the identities of the C-terminus side involving in the host recognition were significantly lower than those of the N-terminus side of the phage body (tail tube) linker site, with an identity of 42% (coverage of 83%) even to the most homologous tail fiber protein of RSJ5 and an identity of 35% (coverage of 60%) to the tail fiber protein of RS-PI-1 (Table 5). Similarly, the identity to the tail fiber protein of RSB2, which does not have the chaperon domain (pfam13884), was significantly low, namely 51% (coverage of 17%).

[0070]

TABLE 5
Top	Protein	Species	Coverage	Identity	Accession
1	putative phage tail fiber	Ralstonia phage RSJ5	83%	42%	YP_009218128.1
	protein
2	hypothetical protein	Ralstonia solanacearum	64%	35%	WP_064049197.1
3	hypothetical protein	Burkholderia ubonensis	61%	37%	KVW40325.1
	WK94_23500
4	hypothetical protein	Burkholderia ubonensis	59%	37%	WP_143135316.1
5	hypothetical protein	Ralstonia solanacearum	88%	31%	WP_049842194.1
6	hypothetical protein	Burkholderia ubonensis	58%	37%	WP_060368006.1
7	hypothetical protein	Burkholderia ubonensis	58%	37%	WP_060063309.1
8	hypothetical protein	Burkholderia ubonensis	58%	37%	WP_059841529.1
9	hypothetical protein	Burkholderia ubonensis	58%	37%	WP_060032179.1
10	hypothetical protein	Burkholderia ubonensis	58%	37%	WP_060163429.1
11	hypothetical protein	Burkholderia ubonensis	58%	37%	WP_059872146.1
12	hypothetical protein	Ralstonia phage RS-PI-1	60%	35%	AQT27772.1

(7) Summary

[0071] From the above results, it was found that bacteriophage RKP181 has the following properties.

• • [0072] RKP181 is classified to the genus 77-like virus of the family Podoviridae (podovirus) of the order Caudovirales.
  • [0073] The genome of RKP181 is linear double-stranded DNA.
  • [0074] The genomic structure is type 17 having a short direct terminal redundant sequence structure.
  • [0075] The genome size is 39,200 bp to 39,500 bp, preferably 39,455 bp (including two terminal redundant sequences) or 39,216 bp (including one terminal redundant sequence).
  • [0076] The ends of the genome may have a terminal redundant sequence of 200 to 250 bp, preferably 239 bp.
  • [0077] The GC content is 62.6%.
  • [0078] The closest related strain of known strains is Ralstonia phage strain phiITL-1, with an identity of 87%. The next closest strain is Ralstonia phage strain RSB2, with an identity of 77%.
  • [0079] The amino acid sequence at the C-terminus of a tail fiber protein including a host recognition site is the amino acid sequence of SEQ ID NO: 1.

Separation and Control of Ralstonia solanacearum:

(1) Separation from Disease Plant

[0080] A stem of a disease plant was cut, and bacterial ooze was collected. The bacterial ooze was appropriately diluted with distilled water. The diluted solution was applied to Modified SMSA medium, and formed colonies was separated onto CPG agar plates (10 g of peptone, 1 g of casamino acid, 5 g of glucose and 1.7% agar per 1 L).

(2) Separation from Soil

[0081] Soil and water were mixed well and left still, and then the supernatant was separated. The separated supernatant was appropriately diluted with water, and the diluted solution was applied to Modified SMSA medium. Formed colonies was separated onto CPG agar plates.

<Modified SMSA Medium (per 1 L)>

[0082]

	Peptone	10	g
	Glucose	5	g
	Casamino acid	1	g
	Agar	18	g
	Bacitracin (10 mg/mL)	2.5	mL
	Polymyxin B sulfate (50 mg/mL)	2	mL
	Chloramphenicol (10 mg/mL)	0.5	mL
	Penicillin G potassium (1 mg/mL)	0.5	mL
	Crystal violet (1 mg/mL)	5	mL
	Tetrazolium chloride (10 mg/mL)	5	mL

(3) Results

[0083] One strain of Ralstonia solanacearum was separated from one field by the above method, and 70 Ralstonia solanacearum strains or more in total from the hosts of the family Solanaceae, the family Zingiberaceae, the family Lamiaceae and the like were separated.

Examination of Host Range of RKP181:

(1) Presence or Absence of Infection

[0084] The presence or the absence of infection was examined by a plaque assay or a spot test.

(2) Plaque Assay

[0085] The Ralstonia solanacearum strains were cultured at 28° C. overnight in CPG medium (10 g of peptone, 1 g of casamino acid and 5 g of glucose per 1 L). The bacterial culture was adjusted to OD₆₀₀ of 0.25 with CPG medium. A serial dilution of a phage solution was prepared. The bacterial solutions and the diluted phage solutions were mixed and left at 28° C. After 30 minutes, the bacterium/phage mixtures were mixed with 3 ml of top agar (3 g of peptone, 0.3 g of casamino acid, 1.7 g of glucose and 5 g of agar per 1 L) and layered onto CPG agar plates. After culturing at 28° C. overnight, the presence or the absence of infection was observed with the presence or the absence of plaques.

(3) Spot Test

[0086] Culture solutions of the Ralstonia solanacearum strains which were cultured at 28° C. overnight in the CPG medium were adjusted to OD₆₀₀ of 0.25 with CPG medium. The bacterial solutions in a volume of 250 μL and 3 ml of top agar were mixed and layered onto CPG agar plates. After top agar was hardened, a phage solution was spotted, and the presence of the absence of plaques was observed to examine the presence or the absence of infection,

(4) Results

[0087] It was found that RKP181 infects all the 100 Ralstonia solanacearum strains or more in total from the hosts of the family Solanaceae, the family Zingiberaceae, the family Lamiaceae and the like separated from the nature, including the 34 strains listed in Table 6, which can be obtained from Genebank Project, NARO, National Agriculture and Food Research Organization (Tsukuba, Ibaraki),

[0088]

TABLE 6
	Bacterial Strain	Source of Separation
	MAFF107624	Tomato
	MAFF211266	Tomato
	MAFF211270	Tomato
	MAFF211543	Tomato
	MAFF301859	Tomato
	MAFF311644	Tomato
	MAFF730103	Tomato
	MAFF730131	Tomato
	MAFF302745	Tomato
	MAFF311632	Tomato
	MAFF211536	Tomato
	MAFF331041	Tomato
	MAFF730139	Eggplant
	MAFF211280	Eggplant
	MAFF211533	Eggplant
	MAFF211468	Chili pepper
	MAFF211516	Bell pepper and sweet green pepper
	MAFF311101	Bell pepper and sweet green pepper
	MAFF311102	Bell pepper and sweet green pepper
	MAFF211479	Ginger
	MAFF211471	Ginger
	MAFF211483	Ginger
	MAFF211484	Ginger
	MAFF211486	Ginger
	MAFF211272	Curcuma
	MAFF211276	Curcuma
	MAFF211278	Curcuma
	MAFF211490	Myoga ginger
	MAFF211492	Myoga ginger
	MAFF211497	Myoga ginger
	MAFF211476	Ginger
	MAFF211414	Potato
	MAFF211429	Potato
	MAFF301558	Potato

Evaluation of Infection Cycle by One-Step Growth Method

[0089] After mixing 990 μL of a culture solution of Ralstonia solanacearum strain MAFF730131 cultured to OD₆₀₀ of 0.15 in CPG medium and 10 μL of a RKP181 phage solution (2×10¹⁰ pfu/mL), the mixture was left still at room temperature to adsorb the bacterium and the phage. After 10 minutes, the mixture was centrifuged at 5,000×g for 10 minutes, and the supernatant was collected. Then, the plaque assay was conducted, and the number of adsorbed phages was calculated. The precipitates were re-suspended in 1 mL of CPG medium, and 150 μL was then added to 29, 850 μL of CPG medium, followed by culturing with shaking at 28° C. For 100 minutes after starting culturing with shaking, a 10 μL sample was taken every 10 minutes and mixed with 990 μL of CPG medium. After mixing, a serial dilution was prepared according to the need, and 10 μL was taken immediately, added to 250 μL of a culture solution of strain MAFF730131 which was adjusted to OD₆₀₀ of 0.22 to 0.24 and stirred. The total volume thereof was added immediately to top agar, mixed and then layered onto CPG medium. The number of formed plaques was counted, and potency was calculated. From the potency and the adsorbed phage numbers at the each time point, the phage number generated in one infection cycle (burst size) was calculated. The results are shown in FIG. 7 and summarized in Table 7.

[0090]

TABLE 7
Latent Period	Rise Period	Infection Cycle	Burst Size
30 minutes	50 minutes	80 minutes	61 ± 7 pfu

[0091] From the above results, it was found that the latent period of RKP181 is 30 minutes and that the infection cycle is 80 minutes. It was also found that the phage number generated in one infection cycle is 6117 pfu.

Genetic Stability:

[0092] RKP181 was amplified by passaging for 10 generations, and seven plaques were isolated. The genomes were prepared after amplifying the isolated seven phages for one more generation, and the genome sizes were determined by the electrophoresis (FIG. 8: left). Moreover, the prepared genomes were treated with Eco81I restriction enzyme. The restriction patterns were examined and observed to be consistent with the matters described in Example 1 above, and thus RKP181 was found to be genetically stable (FIG. 8: right).

Control Test:

[0093] A 1×10⁹ pfu/mL phage solution in a volume of 5 mL was poured to the roots of seedlings on a cell seedling tray (10 seedlings) of a large tomato variety, Sekaiichi. The control group (10 seedlings) was not treated. After six days, the seedlings were planted in pots, and 5 mL of a bacterial culture solution of strain MAFF730131 which was adjusted to OD₆₆₀=0.1 (corresponding to about 1×10⁸ cfu/mL) was poured to the roots of the seedlings. Then, observation was continued for 22 days. The results are shown in Table 8.

[0094]

TABLE 8
	Day 13	Day 20	Day 22
	Control		Control		Control
	Value		Value		Value
Without	Incidence Rate	40	—	50	—	60	—
Treatment	Disease Severity	35		50		60
RKP181	Incidence Rate	20	50	30	40	30	50
Treatment	Disease Severity	20		30		30

[0095] On the 22nd day after the bacterium inoculation, six of the 10 plants withered in the phage-untreated group, and four plants were healthy. On the other hand, in the RKP181 phage-treated group, withering was limited to three of the 10 plants, and seven plants were healthy, with the control value of 50. Thus, the control property of the RKP181 phage was observed.

Preparation of Bacterial Wilt DISEASE Control Agent:

[0096] Bacteriophage RKP181 separated in Example 1 was adjusted to potency of 1×10⁹ pfu/ml, and a bacterial wilt disease control agent was thus obtained.

[0097] The bacteriophage of the invention can be used for controlling bacterial wilt,