WO2017104347A1 - Bacteriophage, bacterial wilt disease control agent, and method for controlling bacterial wilt disease - Google Patents

Bacteriophage, bacterial wilt disease control agent, and method for controlling bacterial wilt disease Download PDF

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WO2017104347A1
WO2017104347A1 PCT/JP2016/084291 JP2016084291W WO2017104347A1 WO 2017104347 A1 WO2017104347 A1 WO 2017104347A1 JP 2016084291 W JP2016084291 W JP 2016084291W WO 2017104347 A1 WO2017104347 A1 WO 2017104347A1
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bacterial wilt
bacteriophage
control agent
rsf1
subunit
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PCT/JP2016/084291
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French (fr)
Japanese (ja)
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山田　隆
藤江　誠
川崎　健
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国立大学法人広島大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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  • the present invention relates to bacteriophage, bacterial wilt control agent and bacterial wilt control method.
  • Bacterial wilt (Ralstonia solanacearum) infects more than 200 kinds of plants including solanaceous plants and causes bacterial wilt that causes the plants to die.
  • the main chemical pesticides that have been used to combat bacterial wilt are the fumigant chloropicrin or methyl bromide.
  • the development of safe alternative pesticides and control techniques to replace chemical pesticides is strongly desired due to problems such as an increase in effective application amount, environmental pollution, ozone layer destruction, health effects and residual pesticides.
  • Patent Document 1 discloses a bacteriophage exhibiting lytic activity in six strains of bacterial wilt.
  • Patent Document 2 discloses linear bacteriophage RSM3 that infects 11 strains including linear bacteriophage RSM1 that infects 6 strains of bacterial wilt and 9 strains that RSM1 does not infect. ing.
  • Patent Document 2 shows that when bacterial wilt fungus infected with RSM1 or RSM3 is pre-inoculated on plants such as tomatoes, bacterial wilt caused by bacterial wilt fungus having strong pathogenicity can be prevented. .
  • Non-Patent Document 1 discloses jumbo phage J6 isolated from Thai soil and infected with bacterial wilt. Pesticides using bacteriophages disclosed in Patent Document 1, Patent Document 2 and Non-Patent Document 1 are promising in terms of higher safety than chemical pesticides such as chloropicrin or methyl bromide.
  • Bunnchoth A 8 others, “Isolation of Ralstonia solanacearum-infecting bacteriophages from tomado fields. In Ching Mai, Thailand, and ther espirem 14 Appl. Microbiol., 118, p. 1023-1033
  • the present invention has been made in view of the above circumstances, and provides a bacteriophage, a bacterial wilt control agent and a bacterial wilt control method capable of controlling bacterial wilt caused by various bacterial wilt bacteria over a longer period of time.
  • the purpose is to do.
  • the bacteriophage according to the first aspect of the present invention is:
  • the genome size is over 200,000 bp
  • the protein constituting the phage particle includes a ⁇ subunit of a virion-related RNA polymerase and a ⁇ ′ subunit of a virion-related RNA polymerase, Infects Ralstonia solanacearum.
  • the genome includes Two or more lytic enzymes are encoded, It is good as well.
  • the bacteriophage is RSF1 indicated by NITE BP-02176, which was deposited on December 10, 2015 at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation for Product Evaluation Technology, It is good as well.
  • the bacterial wilt control agent according to the second aspect of the present invention is:
  • the bacteriophage according to the first aspect of the present invention is included.
  • the bacterial wilt control agent according to the third aspect of the present invention is: The bacteriophage according to the first aspect of the present invention, A bacteriophage that is different from the bacteriophage and infects Ralstonia solanacerum; including.
  • the bacterial wilt control method according to the fourth aspect of the present invention is: The method includes an administration step of administering the bacterial wilt control agent according to the second aspect of the present invention or the bacterial wilt control agent according to the third aspect of the present invention to a plant or a plant growth medium.
  • bacterial wilt caused by various bacterial wilt bacteria can be controlled for a longer period of time.
  • FIG. 1 It is a figure which shows an example of the form of bacteriophage RSF1 which concerns on this invention. It is a figure which shows the size of the genome of RSF1. It is a figure which shows the circular genome map of RSF1. It is a figure which shows the band of SDS-PAGE which isolate
  • the bacteriophage according to the present embodiment has a genome size of 200,000 bp or more and is also called a jumbo phage.
  • the structure of the phage particle of the bacteriophage according to the present embodiment is a myovirus type including a dodecahedron head and a tail.
  • the length of the head is 100 to 130 nm, preferably 110 to 120 nm.
  • the length of the tail is 150 to 200 nm, preferably 170 to 190 nm.
  • the width of the tail is 20 to 30 nm, preferably 22 to 28 nm.
  • the form of RSF1 is shown in FIG.
  • RSF1 was commissioned on December 10, 2015 by the National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818). A request for transfer to an international deposit is received on October 14, 2016 (accession number: NITE BP-02176).
  • the bacteriophage according to the present embodiment is infected with bacterial wilt (Ralstonia solanacerum).
  • the bacteriophage infects a variety of bacterial wilt that differ in race, physiology and phylogenetic type.
  • the bacteriophage has lytic activity against infected bacterial wilt. Examples of bacterial blight fungi strains in which the bacteriophage is effective include C319, M4S, Ps29, Ps65, Ps72, Ps74, RS1002, and the like, and MAFF106603, 106611, 11270 available from the National Institute of Agrobiological Sciences.
  • strains listed here are only examples, and the host range of the bacteriophage according to the present embodiment is considered to be wider.
  • the known Pseudomonas aeruginosa phage KZ includes a virion-related RNA polymerase for expressing genes early in the infection cycle without host RNA polymerase activity, and an early expression RNA polymerase for phage expression in the middle and late stages Multi-subunit RNA polymerase is said to function.
  • the bacteriophage genome according to the present embodiment includes genes corresponding to all of a plurality of genes encoding multi-subunit RNA polymerase possessed by KZ.
  • the plurality of genes encoding the multi-subunit RNA polymerase include two genes encoding the N region and the C region of the ⁇ subunit (RpoB) of virion-related RNA polymerase, respectively, and ⁇ of virion-related RNA polymerase.
  • two genes encoding the N region and C region of the ⁇ subunit (RpoB) of the initial expressed RNA polymerase respectively
  • the initial expression Two genes encoding the N region and C region of the ⁇ ′ subunit (RpoC) of RNA polymerase, respectively.
  • the bacteriophage according to the present embodiment expresses four genes relating to the ⁇ subunit of the virion-related RNA polymerase and the ⁇ ′ subunit of the virion-related RNA polymerase as proteins.
  • the bacteriophage includes a ⁇ -subunit of a virion-related RNA polymerase and a ⁇ ′ subunit of a virion-related RNA polymerase in a protein (virion protein) constituting the phage particle.
  • the bacteriophage according to the present embodiment brings a full set of ⁇ subunit genes and ⁇ ′ subunit genes into phage particles and introduces them into bacterial wilt bacteria together with the genome upon infection with bacterial wilt fungus. . By doing so, the bacteriophage can rapidly and efficiently transcribe genomic DNA without depending on the RNA polymerase of bacterial wilt, and thus exhibits high infection efficiency against bacterial wilt.
  • two or more types of lytic enzymes are encoded in the genome of the bacteriophage according to the present embodiment.
  • the lytic enzyme is, for example, a chitinase-like enzyme, a LysM-like murein lytic enzyme, and a glycosyltransferase. Since the bacteriophage encodes a plurality of types of lytic enzymes in its genome, it can maintain the lytic activity against bacterial wilt for a long time.
  • the bacteriophage is obtained by washing and centrifuging a sample containing soil and filtering the supernatant with a membrane filter. Furthermore, the target bacteriophage can be isolated by using a suitable bacterial wilt fungus as a host. For the isolation of bacteriophage and the measurement of titer, plaque assay is carried out by overlaying a soft agar medium (0.45% agar) with a mixture of bacterial wilt and bacteriophage sample on an agar medium. Is preferred.
  • any method known in the art can be used.
  • a bacteriophage is added to a culture solution containing bacterial wilt cultivated in a CPG medium containing 0.1% casamino acid, 1% peptone and 0.5% glucose, and the bacteriophage is cultivated Can be infected.
  • control includes prevention of bacterial infection by bacterial wilt, prevention of plant disease by bacterial wilt, prevention of plant disease expansion by bacterial wilt and extermination of bacterial wilt.
  • the bacterial wilt control agent includes the bacteriophage.
  • the content of bacteriophage in the bacterial wilt control agent is specified by, for example, infectious units. Infectious units are defined as plaque forming units (pfu), the ability to form transparent areas or plaques on a bacterial culture plate.
  • the bacterial wilt control agent contains, for example, bacteriophage at 10 3 to 10 14 pfu / mL, 10 4 to 10 12 pfu / mL, or 10 3 to 10 8 pfu / mL in a state of being suspended in sterilized water. .
  • the bacterial wilt control agent may contain other substances, compositions and the like that are generally pharmaceutically or botanically acceptable.
  • the bacterial wilt control agent can be used in the bacterial wilt control method.
  • the bacterial wilt control method includes an administration step of administering the bacterial wilt control agent to a plant or a plant growth medium.
  • the plant may be of any kind as long as it can be affected by bacterial wilt disease, but is preferably a solanaceous plant, more specifically tomato, potato, eggplant, tobacco, and the like.
  • the dose of the bacterial wilt control agent in the administration step can be appropriately determined according to the type of plant to be administered or the volume of the plant growth medium.
  • a suitable dose is 10 2 to 10 10 pfu per plant individual, preferably 10 4 to 10 8 pfu.
  • the bacterial wilt control agent contains the above-mentioned dose of bacteriophage in a suitable carrier or diluent, and is 100 ⁇ L to 100 mL or 1 to 10 mL, for example, depending on the type of plant to be administered or the volume of the plant growth medium.
  • the plant growth medium is a structure such as soil, mat, solid medium, nutrient solution in hydroponics, water in hydroponics, and the like.
  • a suspension containing the bacterial wilt control agent is administered to a plant growth medium, for example, 1 ⁇ L to 1000 mL, 10 ⁇ L to 100 mL, 100 ⁇ L to 10 mL, or 1 to 5 mL is sprayed per 1 m 2 of the surface area of the plant growth medium. May be administered in any amount greater than this.
  • the bacterial wilt control agent according to the present embodiment may be administered once to a plant or a plant growth medium, or may be administered multiple times to a plant or a plant growth medium at an arbitrary time interval.
  • the bacterial wilt control agent is administered to plants or the like at intervals of several months, one month or one week, or once every two weeks, once every three weeks, once every four weeks, etc. May be.
  • the administration interval can be appropriately determined according to the plant to be administered or the plant growth medium.
  • the method for administering the bacterial wilt control agent is arbitrary as long as it can expose the plant or the plant growth medium to the bacterial wilt control agent.
  • the method of administering the bacterial wilt control agent includes, for example, spraying and injecting the bacterial wilt control agent, or allowing the bacterial wilt control agent to penetrate into a plant or a plant growth medium. Moreover, you may add a bacterial wilt control agent to the seedling root part of a pot.
  • the control agent When injecting into a plant, the control agent may be put in a syringe and inoculated with pressure, or may be inoculated through an injection needle.
  • the bacterial wilt control agent is administered to a plant by spraying or the like, if the plant is not infected with bacterial wilt, the plant is infected with bacterial wilt or the disease of bacterial wilt is caused to the plant. Can be prevented. If the plant is infected with bacterial wilt, it can be prevented from spreading by administering the bacterial wilt control agent.
  • the bacteriophage contained in the bacterial wilt control agent can be infected with a potential bacterial wilt.
  • bacterial wilt can be controlled.
  • the plant growth medium may be any medium as long as the plant grows.
  • the bacterial wilt control agent can lyse a wide variety of bacterial wilt bacteria. For this reason, the bacterial wilt control agent can control bacterial wilt.
  • the bacteriophage contained in the bacterial wilt control agent according to the present embodiment has the property of specifically infecting various bacterial wilt fungi, so it has high specificity and affects other useful microorganisms. Absent. Thereby, the bacterial wilt control agent can make the influence on the environment as small as possible. Therefore, the bacterial wilt control agent is safer than chemical pesticides, and can avoid problems such as an increase in resistant bacteria, an increase in effective application amount, environmental pollution, residual pesticides, and health effects.
  • the bacteriophage contained in the bacterial wilt control agent according to the present embodiment can control diseases caused by bacterial wilt bacteria over a long period of time as shown in Example 8 below.
  • the bacterial wilt control agent according to the present embodiment is different from the first bacteriophage in addition to the bacteriophage according to the first embodiment (hereinafter referred to as “first bacteriophage”) and A bacteriophage that infects (hereinafter referred to as “second bacteriophage”).
  • first bacteriophage the first bacteriophage
  • second bacteriophage A bacteriophage that infects
  • the second bacteriophage is not particularly limited, but preferably its host range is different from the first bacteriophage. More preferably, as the second bacteriophage, a strain of bacterial wilt that has a relatively low lytic activity by the first bacteriophage exhibits an antibacterial activity higher than that by the first bacteriophage. Preferably, the second bacteriophage is infected with a strain of bacterial wilt that is not infected by the first bacteriophage. The second bacteriophage may have a different infection cycle from the infection cycle of the first bacteriophage.
  • the second bacteriophage is, for example, ⁇ RSM1, which has been entrusted to the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation as NITE BP-1085, and the same as NITE BP-1086.
  • the bacterial wilt control agent according to the present embodiment is not limited to one type of bacteriophage as the second bacteriophage, and may include a plurality of types of bacteriophages.
  • a bacterial wilt control agent containing RSF1 as the first bacteriophage and RSB1 as the second bacteriophage has an effect against bacterial wilt like the bacterial wilt control agent containing only RSF1 as the bacteriophage. Can be controlled.
  • the bacterial wilt control agent containing RSF1 and RSB1 is effective for both bacterial wilt fungi contained in the host range of RSF1 and bacterial wilt fungus contained in the host range of RSB1.
  • the blending ratio of the first bacteriophage and the second bacteriophage in the bacterial wilt control agent according to the present embodiment is not particularly limited and may be set arbitrarily.
  • the bacterial wilt control agent is obtained by suspending the first bacteriophage in a state of 10 3 to 10 14 pfu / mL, 10 4 to 10 12 pfu / mL, or 10 3 to 10 8 pfu / mL in a state of being suspended in sterilized water.
  • a second bacteriophage at 10 3 to 10 14 pfu / mL, 10 4 to 10 12 pfu / mL or 10 3 to 10 8 pfu / mL.
  • the bacterial wilt control agent includes a plurality of types of bacteriophages that are infected with bacterial wilt. By using bacteriophages with different host ranges, a wider variety of bacterial wilt can be lysed. Moreover, various lytic activity characteristics can be obtained by using bacteriophages with different infection cycles.
  • Example 1 Isolation and purification of RSF type jumbo phage
  • the bacterial wilt strain used for the test was a CPG medium containing 0.1% (W / V) casamino acid, 1.0% (W / V) peptone and 0.5% (W / V) glucose. The culture was shaken at 28 ° C. (200 to 300 rpm).
  • the supernatant was filtered with a membrane filter having a membrane pore diameter of 0.45 ⁇ m, and the precipitate was further filtered with SM buffer (50 mmol / L Tris-HCl (pH 7.5), 100 mmol / L NaCl, 10 mmol / L MgSO 4 and 0. (01% gelatin).
  • SM buffer 50 mmol / L Tris-HCl (pH 7.5), 100 mmol / L NaCl, 10 mmol / L MgSO 4 and 0. (01% gelatin).
  • the RSF1 suspension was mixed with CsCl (9.4 g / 20 mL) and 18 hours at 145,000 ⁇ g using the P28S rotor of a CP100 ⁇ ultracentrifuge (Hitachi). Ultracentrifuged. Purified RSF1 was stored at 4 ° C. until use.
  • Example 2 Structure of RSF1 phage particle
  • RSF1 phage particles (10 12 pfu / mL) were negatively stained with phosphotungstic acid and observed with an electron microscope (H600A, manufactured by Hitachi, Ltd.).
  • the RSF1 phage particle was a myovirus type with an icosahedral head of about 115 nm, a tail length of 180 nm, and a tail width of 25 nm.
  • the bar in FIG. 1 indicates a length of 100 nm.
  • Genomic DNA was isolated from phage particles by phenol extraction. To determine the size of the genome, purified phage particles were embedded in 0.5% low melting point agarose (InCert TM agarose, manufactured by FMC). Next, it was treated with 1 mg / mL protease K (manufactured by Merck) and 1% (W / V) Sarkosyl, and the nucleic acid was subjected to pulse field using a CHEF MAPPER TM electrophoresis apparatus (manufactured by Bio-Rad). Gel electrophoresis was performed.
  • InCert TM agarose 0.5% low melting point agarose
  • FIG. 2 shows bands obtained by pulse field gel electrophoresis.
  • Lane 1 shows a lambda ladder band which is a size marker.
  • the genome size of RSF1 shown in lane 2 was about 230 kbp.
  • Example 4 Genomic analysis of RSF1
  • Shotgun sequencing of RSF1 genomic DNA was performed on a GS Junior Sequence System (Roche). Assembly of the determined base sequence was performed with GS De Novo Assembler v2.6. The analyzed base sequence was 222,888 bp.
  • An open reading frame (ORF) greater than 150 bp beginning with “ATG” was identified with Glimmer v3.02.
  • a homology search was performed on the sequence database using BLASTP / RPS-BLAST. In the homology search, E-value was less than 1e-5 as a significant similarity cut-off.
  • sequence databases are KEGG GENES, NCBI / Cdd sequence domain database (version 3.12), UniProt sequence database (Release 2014_08), NCBI Refseq complete 20 .
  • the tRNA genes were identified using tRNAScan-SE 1.4 (option; -B for bacterial tRNAs).
  • the circular genome map was drawn with CGView.
  • FIG. 1 A circular genome map of RSF1 is shown in FIG.
  • the genome was a 222,888 bp double-stranded DNA with circular overlap.
  • a total of 230 genes were encoded in the genome. Of the 230 genes, 55 were encoded clockwise and the rest were encoded counterclockwise. Based on the similarity to proteins that were biologically characterized in the sequence database, 27 ORF annotations could be determined.
  • the N region and C region of the ⁇ subunit (RpoB) of virion-related RNA polymerase were encoded by ORF40 and ORF51, respectively.
  • the N region and C region of the ⁇ 'subunit (RpoC) of virion-related RNA polymerase were encoded by ORF41 and ORF199, respectively.
  • the N region and C region of the ⁇ subunit (RpoB) of the initially expressed RNA polymerase were encoded by ORF122 and ORF215, respectively.
  • the N region and C region of the ⁇ ′ subunit (RpoC) of the early expressed RNA polymerase were encoded by ORF227 and ORF214, respectively.
  • LysM-like murein lytic enzyme (chitinase-like), which is a lytic enzyme in RSF1 genome, was encoded by ORF42.
  • SLT glycosyltransferase which is a lytic enzyme, was encoded by ORF55.
  • ORFs that are homologues of proteins related to DNA replication in the genome of RSF1 include ORF57 (RNase H), ORF63 (SbcC-ATPase), ORF105 (DNA ligase) and ORF126 (DnaB helicase).
  • ORF68 is highly similar to GIY-YIG type nuclease.
  • ORF190 thymidylate kinase
  • ORF164 thymidylate synthase
  • ORF77 and ORF78 dihydrofolate reductase
  • ORF118 ribonucleotide reductase ⁇ subunit
  • ORF117 ribonucleotide reductase ⁇ subunit
  • ORF119 anaerobic ribonucleotide diphosphate reductase
  • ORFs having homology to the host or phage interaction-related protein and the gene encoding the protein constituting the phage particle were identified.
  • Example 5 Identification of structural protein by nanoLC-EIS MS / MS
  • SDS-PAGE 10-12% (W / V) polyacrylamide
  • Protein bands were visualized by staining the gel with Coomassie Brilliant Blue, excised from the gel, reduced with dithiothreitol, alkylated with iodoacetamide, and then fragmented with trypsin.
  • the trypsin peptide was trapped using a short ODS column (PepMap 100; 5 ⁇ m C18, 5 mm ⁇ 300 ⁇ m ID, manufactured by Thermo Fisher Scientific), and ODS column (Nano HPLC Capillary Column, 3 ⁇ 75 C18, 3 ⁇ m C18mm
  • the product was separated using a nano-liquid chromatography (nanoLC).
  • nanoLC For nanoLC, Ultimate TM 3000 RSLC nano system (manufactured by Thermo Fisher Scientific) was used.
  • the mobile phases in the separation were solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile).
  • solvent A 0.1% formic acid
  • solvent B 0.1% formic acid in acetonitrile
  • the concentrated tryptic peptide was eluted from the trap column and a series of isocratic and linear gradient (0 ⁇ 3 minutes solvent A, 3 ⁇ 35 minutes 0-35% (v / v) solvent B, and 10 minutes increase to 90% solvent B and 15 minutes re-equilibration with solvent A) Separation with a separation column.
  • MS spectra and MS / MS spectra were obtained in positive ion mode using Orbitrap (mass range: m / z 300-1500) and Iontrap (scanning dependent data of top 5 peaks using CID), respectively.
  • the capillary source voltage was set to 1.5 kV and the transfer capillary temperature was maintained at 200 ° C.
  • the capillary voltage and tube lens voltage were 20 V and 80 V, respectively.
  • Assignment of MS / MS data to the tryptic peptide encoded by the ORF of RSF1 was performed using the Xcalibur program (ver2.0, manufactured by Thermo Fisher Scientific).
  • FIG. 4 shows the SDS-PAGE band from which the structural protein of RSF1 was separated.
  • Proteins constituting RSF1 phage particles included proteins derived from ORF40, ORF51, ORF41, and ORF199.
  • RSL2 structural protein of jumbo phage J6
  • RSL2 has a genome size of about 220 kbp
  • the phage particle is a myovirus type like RSF1.
  • the structural proteins constituting RSL2 phage particles include ORF37 and ORF48-derived proteins encoding the N and C regions of the ⁇ subunit of virion-related RNA polymerase, respectively, and ⁇ ′ subunit A protein derived from ORF192 encoding the C region was detected, but a protein derived from ORF38 encoding the N region of the ⁇ ′ subunit was not detected.
  • Example 6 Examination of host range of RSF1 The host range of RSF1 was examined by the above plaque assay using various bacterial wilt strains. Furthermore, the host range of RSL2 was also examined for comparison.
  • Table 1 shows the host range of RSL2 and RSF1.
  • sensitivity to RSL2 or RSF1
  • “+” indicates sensitivity
  • ⁇ indicates insensitivity (resistance).
  • RSF1 infects 19 strains of various plants as host plants.
  • the strains infected with RSF1 include Ps65, MAFF21114, MAFF301485 and MAFF301558, which are not infected with RSL2, and RSF1 showed a broad host range for bacterial wilt strains derived from Japan compared to RSL2. .
  • RSF1 only infects bacterial wilt and does not infect enterobacteria, Pseudomonas, Rhizobium, Gram-positive bacteria, and the like.
  • Example 7 Evaluation of infection cycle of RSF1
  • the infection cycle was evaluated by a one-step growth method.
  • the MAFF730138 strain having an OD600 of 0.1 by culture was collected by centrifugation (6000 ⁇ g) and suspended in CPG medium so that the final culture volume was 10 mL (approximately 1 ⁇ 10 8 cfu / mL).
  • RSF1 The time course of the number of RSF1 per cell is shown in FIG.
  • RSF1 had an incubation period of 90 minutes and an infection cycle of 4 hours, and the burst size was about 80 pfu / cell.
  • the burst size was about 40-50 pfu / cell with an incubation period of 150 minutes and 4.5 hours of incubation period.
  • RSF2 relies on the host RNA polymerase for early expression because RSL2 lacks part of the ⁇ ′ subunit of the virion-related RNA polymerase in the phage particle, whereas RSF1 relies on the ⁇ -subunit of the virion-related RNA polymerase in the phage particle. Because it has a full set of units and ⁇ ′ subunits, the incubation period and infection cycle are considered short. Thereby, the infection efficiency of RSF1 is higher than RSL2.
  • Example 8 Evaluation of bacterial wilt control effect of RSF1 in tomato
  • FIGS. 7 (A) and (B) show the symptom appearing in the tomato seedlings in the control group and the phage-treated group, respectively.
  • Symptom index “0” is unchanged, “1” is upward from the cotyledon, the first leaf is wilt, “2” is wilt the second leaf, “3” is wilt the third leaf, “4” is the first Four leaves indicate wilting and “5” indicates death.
  • FIG. 7 (A) a remarkable symptom of bacterial wilt appeared about 1 week after administration of the cell suspension in the tomato seedlings in the control group. After about 2 weeks, 80% of the tomato seedlings in the control group died.
  • FIG. 7 (A) shows a remarkable symptom of bacterial wilt appeared about 1 week after administration of the cell suspension in the tomato seedlings in the control group. After about 2 weeks, 80% of the tomato seedlings in the control group died.
  • FIG. 7 (A) shows a remarkable symptom of bacterial w
  • the tomato seedlings did not change even after 3 weeks in the phage-treated group. Thereafter, the first leaves slightly withered were observed in the tomato seedlings in the phage-treated group, which was different from the symptoms of bacterial wilt. The tomato seedlings in the phage-treated area did not develop bacterial wilt even after one month. Moreover, it is thought that the resistant microbe to RSF1 which generate
  • the present invention is suitable for controlling bacterial wilt, preventing spreading, or controlling bacterial wilt.

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Abstract

A bacteriophage having a genome size of 200,000 bp or longer, wherein a protein constituting phage particles of the bacteriophage contains both of a β-subunit of virion-associated RNA polymerase and a β'-subunit of virion-associated RNA polymerase. The bacteriophage can be transmitted to Ralstonia solanacearum.

Description

バクテリオファージ、青枯病防除剤及び青枯病防除方法Bacteriophage, bacterial wilt control agent and bacterial wilt control method
　本発明は、バクテリオファージ、青枯病防除剤及び青枯病防除方法に関する。 The present invention relates to bacteriophage, bacterial wilt control agent and bacterial wilt control method.
　青枯病菌（Ｒａｌｓｔｏｎｉａ　ｓｏｌａｎａｃｅａｒｕｍ）はナス科植物をはじめ２００種以上の植物に感染し、該植物を枯死させる青枯病を引き起こす。青枯病の対策に使用されてきた主な化学農薬は劇物である燻蒸剤クロロピクリン又は臭化メチルである。しかし、有効散布量の増大、環境汚染、オゾン層破壊、健康への影響及び残留農薬などの問題から、化学農薬に代わる安全な代替農薬及び防除技術の開発が強く望まれている。 Bacterial wilt (Ralstonia solanacearum) infects more than 200 kinds of plants including solanaceous plants and causes bacterial wilt that causes the plants to die. The main chemical pesticides that have been used to combat bacterial wilt are the fumigant chloropicrin or methyl bromide. However, the development of safe alternative pesticides and control techniques to replace chemical pesticides is strongly desired due to problems such as an increase in effective application amount, environmental pollution, ozone layer destruction, health effects and residual pesticides.
　特許文献１には、青枯病菌の６種類の株に溶菌活性を示すバクテリオファージが開示されている。特許文献２には、青枯病菌の６種類の株に感染する線状のバクテリオファージＲＳＭ１及びＲＳＭ１が感染しない９種類の株を含む１１種類の株に感染する線状のバクテリオファージＲＳＭ３が開示されている。特許文献２では、ＲＳＭ１又はＲＳＭ３を感染させた青枯病菌をトマト等の植物に予め接種しておくと、強病原性を有する青枯病菌による青枯病の発症を予防できることが示されている。 Patent Document 1 discloses a bacteriophage exhibiting lytic activity in six strains of bacterial wilt. Patent Document 2 discloses linear bacteriophage RSM3 that infects 11 strains including linear bacteriophage RSM1 that infects 6 strains of bacterial wilt and 9 strains that RSM1 does not infect. ing. Patent Document 2 shows that when bacterial wilt fungus infected with RSM1 or RSM3 is pre-inoculated on plants such as tomatoes, bacterial wilt caused by bacterial wilt fungus having strong pathogenicity can be prevented. .
　さらに、非特許文献１には、タイの土壌から単離された、青枯病菌に感染するジャンボファージＪ６が開示されている。特許文献１、特許文献２及び非特許文献１に開示されたバクテリオファージを使用する農薬は、クロロピクリン又は臭化メチルなどの化学農薬よりは安全性が高い点で有望である。 Furthermore, Non-Patent Document 1 discloses jumbo phage J6 isolated from Thai soil and infected with bacterial wilt. Pesticides using bacteriophages disclosed in Patent Document 1, Patent Document 2 and Non-Patent Document 1 are promising in terms of higher safety than chemical pesticides such as chloropicrin or methyl bromide.
特開２００５－２７８５１３号公報JP 2005-278513 A 特開２０１２－２３１７３１号公報JP 2012-231731 A
　青枯病菌には、宿主域によるレース（ｒａｃｅ）、糖類の代謝型による生理型（ｂｉｏｖａｒ）及び遺伝子情報による系統型（ｐｈｙｌｏｔｙｐｅ）の異なる様々な株が存在する。世界各地で発生し得る青枯病の病害を防ぐために、さらに多様な青枯病菌に有効な新規のバクテリオファージが求められている。また、使用頻度を抑えることで利便性を高め、かつコストを削減するために、長期に渡って防除効果を発揮するバクテリオファージが望ましい。 There are various strains of bacterial wilt that have different races based on the host range, physiological types based on saccharide metabolism, and phylotypes based on genetic information. In order to prevent bacterial wilt disease that can occur in various parts of the world, a new bacteriophage effective against various bacterial wilt fungi is required. In addition, a bacteriophage that exerts a control effect over a long period of time is desirable in order to improve convenience by reducing the frequency of use and to reduce costs.
　本発明は、上記実情に鑑みてなされたものであり、多様な青枯病菌に起因する青枯病をより長期に渡って防除できるバクテリオファージ、青枯病防除剤及び青枯病防除方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and provides a bacteriophage, a bacterial wilt control agent and a bacterial wilt control method capable of controlling bacterial wilt caused by various bacterial wilt bacteria over a longer period of time. The purpose is to do.
　本発明の第１の観点に係るバクテリオファージは、
　ゲノムのサイズが２００，０００ｂｐ以上であって、
　ファージ粒子を構成するタンパク質に、ビリオン関連ＲＮＡポリメラーゼのβサブユニットとビリオン関連ＲＮＡポリメラーゼのβ’サブユニットとを含み、
　Ｒａｌｓｔｏｎｉａ　ｓｏｌａｎａｃｅａｒｕｍに感染する。 The bacteriophage according to the first aspect of the present invention is:
The genome size is over 200,000 bp,
The protein constituting the phage particle includes a β subunit of a virion-related RNA polymerase and a β ′ subunit of a virion-related RNA polymerase,
Infects Ralstonia solanacearum.
　この場合、前記ゲノムには、
　２種類以上の溶菌酵素がコードされている、
　こととしてもよい。 In this case, the genome includes
Two or more lytic enzymes are encoded,
It is good as well.
　また、上記バクテリオファージは、
　独立行政法人製品評価技術基盤機構特許微生物寄託センターに２０１５年１２月１０日に受託された受託番号：ＮＩＴＥ　ＢＰ－０２１７６で示されるＲＳＦ１である、
　こととしてもよい。 In addition, the bacteriophage is
RSF1 indicated by NITE BP-02176, which was deposited on December 10, 2015 at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation for Product Evaluation Technology,
It is good as well.
　本発明の第２の観点に係る青枯病防除剤は、
　上記本発明の第１の観点に係るバクテリオファージを含む。 The bacterial wilt control agent according to the second aspect of the present invention is:
The bacteriophage according to the first aspect of the present invention is included.
　本発明の第３の観点に係る青枯病防除剤は、
　上記本発明の第１の観点に係るバクテリオファージと、
　前記バクテリオファージと異なり、かつＲａｌｓｔｏｎｉａ　ｓｏｌａｎａｃｅａｒｕｍに感染するバクテリオファージと、
　を含む。 The bacterial wilt control agent according to the third aspect of the present invention is:
The bacteriophage according to the first aspect of the present invention,
A bacteriophage that is different from the bacteriophage and infects Ralstonia solanacerum;
including.
　本発明の第４の観点に係る青枯病防除方法は、
　上記本発明の第２の観点に係る青枯病防除剤又は上記本発明の第３の観点に係る青枯病防除剤を、植物又は植物成長媒体に投与する投与ステップを含む。 The bacterial wilt control method according to the fourth aspect of the present invention is:
The method includes an administration step of administering the bacterial wilt control agent according to the second aspect of the present invention or the bacterial wilt control agent according to the third aspect of the present invention to a plant or a plant growth medium.
　本発明によれば、多様な青枯病菌に起因する青枯病をより長期に渡って防除できる。 According to the present invention, bacterial wilt caused by various bacterial wilt bacteria can be controlled for a longer period of time.
本発明に係るバクテリオファージＲＳＦ１の形態の一例を示す図である。It is a figure which shows an example of the form of bacteriophage RSF1 which concerns on this invention. ＲＳＦ１のゲノムのサイズを示す図である。It is a figure which shows the size of the genome of RSF1. ＲＳＦ１の環状ゲノム地図を示す図である。It is a figure which shows the circular genome map of RSF1. ＲＳＦ１のファージ粒子のタンパク質を分離したＳＤＳ－ＰＡＧＥのバンドを示す図である。It is a figure which shows the band of SDS-PAGE which isolate | separated the protein of the phage particle | grains of RSF1. ＲＳＬ２のファージ粒子のタンパク質を分離したＳＤＳ－ＰＡＧＥのバンドを示す図である。It is a figure which shows the band of SDS-PAGE which isolate | separated the protein of the phage particle | grains of RSL2. ＲＳＦ１の感染サイクルを示す図である。It is a figure which shows the infection cycle of RSF1. ＲＳＦ１による青枯病防除効果を示す図である。（Ａ）は対照区の結果を示す図である。（Ｂ）はファージ処理区の結果を示す図である。It is a figure which shows the bacterial wilt disease control effect by RSF1. (A) is a figure which shows the result of a control section. (B) is a figure which shows the result of a phage processing section.
　本発明に係る実施の形態について添付の図面を参照して説明する。なお、本発明は下記の実施の形態及び図面によって限定されるものではない。 Embodiments according to the present invention will be described with reference to the accompanying drawings. In addition, this invention is not limited by the following embodiment and drawing.
　（実施の形態１）
　本実施の形態に係るバクテリオファージは、ゲノムのサイズが２００，０００ｂｐ以上でジャンボファージとも言われる。本実施の形態に係るバクテリオファージのファージ粒子の構造は、十二面体の頭部と尾部とを含むｍｙｏｖｉｒｕｓ型である。頭部の長さは１００～１３０ｎｍ、好ましくは１１０～１２０ｎｍである。尾部の長さは１５０～２００ｎｍ、好ましくは１７０～１９０ｎｍである。尾部の幅は２０～３０ｎｍ、好ましくは２２～２８ｎｍである。当該バクテリオファージの一例として、図１にＲＳＦ１の形態を示す。ＲＳＦ１は、独立行政法人製品評価技術基盤機構特許微生物寄託センター（〒２９２－０８１８　日本国千葉県木更津市かずさ鎌足２－５－８　１２２号室）に２０１５年１２月１０日に受託され、ブタペスト条約に基づく国際寄託への移管請求が２０１６年１０月１４日に受領されている（受託番号：ＮＩＴＥ　ＢＰ－０２１７６）。 (Embodiment 1)
The bacteriophage according to the present embodiment has a genome size of 200,000 bp or more and is also called a jumbo phage. The structure of the phage particle of the bacteriophage according to the present embodiment is a myovirus type including a dodecahedron head and a tail. The length of the head is 100 to 130 nm, preferably 110 to 120 nm. The length of the tail is 150 to 200 nm, preferably 170 to 190 nm. The width of the tail is 20 to 30 nm, preferably 22 to 28 nm. As an example of the bacteriophage, the form of RSF1 is shown in FIG. RSF1 was commissioned on December 10, 2015 by the National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818). A request for transfer to an international deposit is received on October 14, 2016 (accession number: NITE BP-02176).
　本実施の形態に係るバクテリオファージは、青枯病菌（Ｒａｌｓｔｏｎｉａ　ｓｏｌａｎａｃｅａｒｕｍ）に感染する。当該バクテリオファージは、レース、生理型及び系統型が異なる多様な青枯病菌に感染する。当該バクテリオファージは、感染した青枯病菌に対する溶菌活性を有する。当該バクテリオファージが有効な青枯病菌の株としては、例えば、Ｃ３１９、Ｍ４Ｓ、Ｐｓ２９、Ｐｓ６５、Ｐｓ７２、Ｐｓ７４、ＲＳ１００２などに加え、独立行政法人農業生物資源研究所から入手可能なＭＡＦＦ１０６６０３、１０６６１１、２１１２７０、２１１５１４、３０１４８５、３０１５５６、３０１５５８、３２７０３２、７３０１０３、７３０１３５、７３０１３８、７３０１３９などが挙げられる。なお、ここに挙げた株は例示であって、本実施の形態に係るバクテリオファージの宿主域は、さらに広いと考えられる。 The bacteriophage according to the present embodiment is infected with bacterial wilt (Ralstonia solanacerum). The bacteriophage infects a variety of bacterial wilt that differ in race, physiology and phylogenetic type. The bacteriophage has lytic activity against infected bacterial wilt. Examples of bacterial blight fungi strains in which the bacteriophage is effective include C319, M4S, Ps29, Ps65, Ps72, Ps74, RS1002, and the like, and MAFF106603, 106611, 11270 available from the National Institute of Agrobiological Sciences. 21514, 301485, 301556, 301558, 327032, 730103, 730135, 730138, 730139 and the like. The strains listed here are only examples, and the host range of the bacteriophage according to the present embodiment is considered to be wider.
　公知のＰｓｅｕｄｏｍｏｎａｓ　ａｅｒｕｇｉｎｏｓａファージＫＺでは、感染サイクルにおいて、宿主のＲＮＡポリメラーゼの活性がない初期に遺伝子を発現させるためのビリオン関連ＲＮＡポリメラーゼと、中期及び後期におけるファージ発現のための初期発現ＲＮＡポリメラーゼとを含むマルチサブユニットＲＮＡポリメラーゼが機能すると言われている。本実施の形態に係るバクテリオファージのゲノムには、ＫＺが有するマルチサブユニットＲＮＡポリメラーゼをコードする複数の遺伝子すべてに対応する遺伝子が含まれている。 The known Pseudomonas aeruginosa phage KZ includes a virion-related RNA polymerase for expressing genes early in the infection cycle without host RNA polymerase activity, and an early expression RNA polymerase for phage expression in the middle and late stages Multi-subunit RNA polymerase is said to function. The bacteriophage genome according to the present embodiment includes genes corresponding to all of a plurality of genes encoding multi-subunit RNA polymerase possessed by KZ.
　より詳細には、マルチサブユニットＲＮＡポリメラーゼをコードする複数の遺伝子は、ビリオン関連ＲＮＡポリメラーゼのβサブユニット（ＲｐｏＢ）のＮ領域及びＣ領域をそれぞれコードする２個の遺伝子、ビリオン関連ＲＮＡポリメラーゼのβ’サブユニット（ＲｐｏＣ）のＮ領域及びＣ領域をそれぞれコードする２個の遺伝子、初期発現ＲＮＡポリメラーゼのβサブユニット（ＲｐｏＢ）のＮ領域及びＣ領域をそれぞれコードする２個の遺伝子、及び初期発現ＲＮＡポリメラーゼのβ’サブユニット（ＲｐｏＣ）のＮ領域及びＣ領域をそれぞれコードする２個の遺伝子である。 More specifically, the plurality of genes encoding the multi-subunit RNA polymerase include two genes encoding the N region and the C region of the β subunit (RpoB) of virion-related RNA polymerase, respectively, and β of virion-related RNA polymerase. 'Two genes encoding the N region and C region of the subunit (RpoC), two genes encoding the N region and C region of the β subunit (RpoB) of the initial expressed RNA polymerase, respectively, and the initial expression Two genes encoding the N region and C region of the β ′ subunit (RpoC) of RNA polymerase, respectively.
　実際、本実施の形態に係るバクテリオファージは、上記のビリオン関連ＲＮＡポリメラーゼのβサブユニット及びビリオン関連ＲＮＡポリメラーゼのβ’サブユニットに関する４個の遺伝子をタンパク質として発現させる。このため、当該バクテリオファージは、ファージ粒子を構成するタンパク質（ビリオンタンパク質）に、ビリオン関連ＲＮＡポリメラーゼのβサブユニットとビリオン関連ＲＮＡポリメラーゼのβ’サブユニットとを含む。本実施の形態に係るバクテリオファージは、フルセットのβサブユニットの遺伝子及びβ’サブユニットの遺伝子をファージ粒子内に持ち込み、青枯病菌への感染時にゲノムと一緒に青枯病菌内に導入する。こうすることで、当該バクテリオファージは、青枯病菌のＲＮＡポリメラーゼに依存せずに迅速かつ効率的にゲノムＤＮＡを転写できるので、青枯病菌への高い感染効率を示す。 Actually, the bacteriophage according to the present embodiment expresses four genes relating to the β subunit of the virion-related RNA polymerase and the β ′ subunit of the virion-related RNA polymerase as proteins. For this reason, the bacteriophage includes a β-subunit of a virion-related RNA polymerase and a β ′ subunit of a virion-related RNA polymerase in a protein (virion protein) constituting the phage particle. The bacteriophage according to the present embodiment brings a full set of β subunit genes and β ′ subunit genes into phage particles and introduces them into bacterial wilt bacteria together with the genome upon infection with bacterial wilt fungus. . By doing so, the bacteriophage can rapidly and efficiently transcribe genomic DNA without depending on the RNA polymerase of bacterial wilt, and thus exhibits high infection efficiency against bacterial wilt.
　好ましくは、本実施の形態に係るバクテリオファージのゲノムには、２種類以上の溶菌酵素がコードされている。溶菌酵素は、例えば、キチナーゼ様酵素、ＬｙｓＭ様ムレイン溶解酵素及び糖転移酵素である。当該バクテリオファージは、複数の種類の溶菌酵素をゲノムにコードしているため、長期に渡って青枯病菌に対する溶菌活性を維持できる。 Preferably, two or more types of lytic enzymes are encoded in the genome of the bacteriophage according to the present embodiment. The lytic enzyme is, for example, a chitinase-like enzyme, a LysM-like murein lytic enzyme, and a glycosyltransferase. Since the bacteriophage encodes a plurality of types of lytic enzymes in its genome, it can maintain the lytic activity against bacterial wilt for a long time.
　上記バクテリオファージは、土壌などを含む試料を、洗浄して遠心分離し、膜フィルターで上清を濾過することで得られる。さらに、宿主として適切な青枯病菌を用いることで、目的とするバクテリオファージを単離することができる。バクテリオファージの単離及び力価の測定には、寒天培地に青枯病菌とバクテリオファージ試料との混合液を加えた軟寒天培地（０．４５％寒天）を重層してプラークを形成させるプラークアッセイが好適である。 The bacteriophage is obtained by washing and centrifuging a sample containing soil and filtering the supernatant with a membrane filter. Furthermore, the target bacteriophage can be isolated by using a suitable bacterial wilt fungus as a host. For the isolation of bacteriophage and the measurement of titer, plaque assay is carried out by overlaying a soft agar medium (0.45% agar) with a mixture of bacterial wilt and bacteriophage sample on an agar medium. Is preferred.
　バクテリオファージの青枯病菌への感染方法は、当該技術分野で公知である任意の方法を用いることができる。一例としては０．１％カザミノ酸、１％ペプトン及び０．５％グルコースを含有するＣＰＧ培地で培養した青枯病菌を含む培養液にバクテリオファージを加え、培養することでバクテリオファージを青枯病菌に感染させることができる。 As a method for infecting bacteriophage with bacterial wilt, any method known in the art can be used. As an example, a bacteriophage is added to a culture solution containing bacterial wilt cultivated in a CPG medium containing 0.1% casamino acid, 1% peptone and 0.5% glucose, and the bacteriophage is cultivated Can be infected.
　上記バクテリオファージは、下記実施例１に示すように幅広い宿主域を有しており、感染した青枯病菌を溶菌する。このため、当該バクテリオファージは、青枯病防除剤の有効成分として好適である。ここで、「防除」とは、青枯病菌の植物への感染の予防、青枯病菌による植物の病害の予防、青枯病菌による植物の病害の拡大防止及び青枯病菌の駆除を含む。 The bacteriophage has a wide host range as shown in Example 1 below, and lyses infected bacterial wilt. Therefore, the bacteriophage is suitable as an active ingredient of the bacterial wilt control agent. Here, “control” includes prevention of bacterial infection by bacterial wilt, prevention of plant disease by bacterial wilt, prevention of plant disease expansion by bacterial wilt and extermination of bacterial wilt.
　本実施の形態に係る青枯病防除剤は、上記バクテリオファージを含む。青枯病防除剤におけるバクテリオファージの含有量は、例えば感染単位で特定される。感染単位は細菌培養用平板上に透明領域又はプラークを形成する能力であるプラーク形成単位（ｐｆｕ）として定義される。青枯病防除剤は、例えば、滅菌水に懸濁した状態で、バクテリオファージを、１０３～１０１４ｐｆｕ／ｍＬ、１０４～１０１２ｐｆｕ／ｍＬ又は１０３～１０８ｐｆｕ／ｍＬで含む。青枯病防除剤は、有効成分であるバクテリオファージ以外にも、一般に薬学的又は植物学的に許容される他の物質、組成物などを含んでもよい。 The bacterial wilt control agent according to the present embodiment includes the bacteriophage. The content of bacteriophage in the bacterial wilt control agent is specified by, for example, infectious units. Infectious units are defined as plaque forming units (pfu), the ability to form transparent areas or plaques on a bacterial culture plate. The bacterial wilt control agent contains, for example, bacteriophage at 10 3 to 10 14 pfu / mL, 10 4 to 10 12 pfu / mL, or 10 3 to 10 8 pfu / mL in a state of being suspended in sterilized water. . In addition to bacteriophage, which is an active ingredient, the bacterial wilt control agent may contain other substances, compositions and the like that are generally pharmaceutically or botanically acceptable.
　当該青枯病防除剤は、青枯病防除方法に利用できる。青枯病防除方法は、上記青枯病防除剤を、植物又は植物成長媒体に投与する投与ステップを含む。植物は、青枯病菌の病害が及び得るものであれば任意の種類であってよいが、好ましくはナス科の植物、より具体的には、トマト、ジャガイモ、ナス及びタバコなどである。 The bacterial wilt control agent can be used in the bacterial wilt control method. The bacterial wilt control method includes an administration step of administering the bacterial wilt control agent to a plant or a plant growth medium. The plant may be of any kind as long as it can be affected by bacterial wilt disease, but is preferably a solanaceous plant, more specifically tomato, potato, eggplant, tobacco, and the like.
　上記投与ステップにおける青枯病防除剤の用量は、投与対象の植物の種類あるいは植物成長媒体の容積などに応じて、適宜决定することができる。例えば、好適な用量は、植物個体あたり１０２～１０１０ｐｆｕ、好ましくは１０４～１０８ｐｆｕである。青枯病防除剤は、上記用量のバクテリオファージを好適な担体又は希釈剤中に含み、投与対象の植物の種類あるいは植物成長媒体の容積などに応じて、例えば１００μＬ～１００ｍＬ又は１～１０ｍＬである。なお、植物成長媒体は、土壌、マット、固形培地などの構造体、養液栽培における養液、及び水栽培における水などである。 The dose of the bacterial wilt control agent in the administration step can be appropriately determined according to the type of plant to be administered or the volume of the plant growth medium. For example, a suitable dose is 10 2 to 10 10 pfu per plant individual, preferably 10 4 to 10 8 pfu. The bacterial wilt control agent contains the above-mentioned dose of bacteriophage in a suitable carrier or diluent, and is 100 μL to 100 mL or 1 to 10 mL, for example, depending on the type of plant to be administered or the volume of the plant growth medium. . The plant growth medium is a structure such as soil, mat, solid medium, nutrient solution in hydroponics, water in hydroponics, and the like.
　当該青枯病防除剤を含む懸濁液を植物成長媒体に投与する場合、例えば、植物成長媒体の表面積１ｍ２あたり、１μＬ～１０００ｍＬ、１０μＬ～１００ｍＬ、１００μＬ～１０ｍＬ、１～５ｍＬを散布することで投与してもよく、これ以上の任意の量で投与してもよい。 When a suspension containing the bacterial wilt control agent is administered to a plant growth medium, for example, 1 μL to 1000 mL, 10 μL to 100 mL, 100 μL to 10 mL, or 1 to 5 mL is sprayed per 1 m 2 of the surface area of the plant growth medium. May be administered in any amount greater than this.
　本実施の形態に係る青枯病防除剤は、植物又は植物成長媒体に単回で投与されてもよいし、任意の時間間隔で植物又は植物成長媒体に複数回投与されてもよい。例えば、青枯病防除剤は、数ヶ月、１ヶ月若しくは１週間に１回又は複数回、あるいは２週間に１回、３週間に１回、４週間に１回などの間隔で植物などに投与されてもよい。投与間隔は、投与対象の植物又は植物成長媒体に応じて、適宜決定することができる。 The bacterial wilt control agent according to the present embodiment may be administered once to a plant or a plant growth medium, or may be administered multiple times to a plant or a plant growth medium at an arbitrary time interval. For example, the bacterial wilt control agent is administered to plants or the like at intervals of several months, one month or one week, or once every two weeks, once every three weeks, once every four weeks, etc. May be. The administration interval can be appropriately determined according to the plant to be administered or the plant growth medium.
　青枯病防除剤を投与する方法は、植物又は植物成長媒体を、当該青枯病防除剤に暴露することができる方法であれば任意である。青枯病防除剤を投与する方法は、例えば、青枯病防除剤を噴霧、注射すること、あるいは当該青枯病防除剤を植物又は植物成長媒体に浸透させることなどである。また、青枯病防除剤をポットの苗根部に添加してもよい。植物に注射する場合、当該防除剤を注射筒に入れ、圧迫接種してもよいし、注射針を介して接種してもよい。 The method for administering the bacterial wilt control agent is arbitrary as long as it can expose the plant or the plant growth medium to the bacterial wilt control agent. The method of administering the bacterial wilt control agent includes, for example, spraying and injecting the bacterial wilt control agent, or allowing the bacterial wilt control agent to penetrate into a plant or a plant growth medium. Moreover, you may add a bacterial wilt control agent to the seedling root part of a pot. When injecting into a plant, the control agent may be put in a syringe and inoculated with pressure, or may be inoculated through an injection needle.
　当該青枯病防除剤を、噴霧などにより植物に投与することで、青枯病菌に未感染の植物であれば、該植物への青枯病菌の感染、あるいは青枯病菌の病害が該植物に及ぶことを防止できる。青枯病に感染後の植物であれば、当該青枯病防除剤を投与することで病害の拡大を阻止できる。 If the bacterial wilt control agent is administered to a plant by spraying or the like, if the plant is not infected with bacterial wilt, the plant is infected with bacterial wilt or the disease of bacterial wilt is caused to the plant. Can be prevented. If the plant is infected with bacterial wilt, it can be prevented from spreading by administering the bacterial wilt control agent.
　青枯病防除剤を植物成長媒体に投与することで、青枯病防除剤に含まれるバクテリオファージを、潜在的な青枯病菌に感染させることができる。この結果、青枯病を防除できる。なお、植物成長媒体は、植物が成長する媒体であれば任意のものでよい。 By administering the bacterial wilt control agent to the plant growth medium, the bacteriophage contained in the bacterial wilt control agent can be infected with a potential bacterial wilt. As a result, bacterial wilt can be controlled. The plant growth medium may be any medium as long as the plant grows.
　以上詳細に説明したように、本実施の形態に係る青枯病防除剤は、幅広い青枯病菌を溶菌させることができる。このため、当該青枯病防除剤は、青枯病を防除できる。 As described in detail above, the bacterial wilt control agent according to the present embodiment can lyse a wide variety of bacterial wilt bacteria. For this reason, the bacterial wilt control agent can control bacterial wilt.
　なお、本実施の形態に係る青枯病防除剤に含まれるバクテリオファージは、種々の青枯病菌に特異的に感染する性質を有するため、特異性が高く、他の有用な微生物に影響を与えない。これにより、当該青枯病防除剤は、環境への影響を極力小さくすることができる。したがって、当該青枯病防除剤は、化学農薬よりも安全性が高く、耐性菌の増加、有効散布量の増大、環境汚染、残留農薬、健康への影響などの問題を回避できる。 Note that the bacteriophage contained in the bacterial wilt control agent according to the present embodiment has the property of specifically infecting various bacterial wilt fungi, so it has high specificity and affects other useful microorganisms. Absent. Thereby, the bacterial wilt control agent can make the influence on the environment as small as possible. Therefore, the bacterial wilt control agent is safer than chemical pesticides, and can avoid problems such as an increase in resistant bacteria, an increase in effective application amount, environmental pollution, residual pesticides, and health effects.
　また、本実施の形態に係る青枯病防除剤に含まれるバクテリオファージは、下記実施例８に示すように、長期に渡って青枯病菌による病害を防除することができる。 Also, the bacteriophage contained in the bacterial wilt control agent according to the present embodiment can control diseases caused by bacterial wilt bacteria over a long period of time as shown in Example 8 below.
　（実施の形態２）
　本実施の形態に係る青枯病防除剤は、上記実施の形態１に係るバクテリオファージ（以下、「第１のバクテリオファージ」とする）に加え、第１のバクテリオファージと異なり、かつ青枯病菌に感染するバクテリオファージ（以下、「第２のバクテリオファージ」とする）をさらに含む。以下、本実施の形態について、上記実施の形態１と異なる点について主に説明する。 (Embodiment 2)
The bacterial wilt control agent according to the present embodiment is different from the first bacteriophage in addition to the bacteriophage according to the first embodiment (hereinafter referred to as “first bacteriophage”) and A bacteriophage that infects (hereinafter referred to as “second bacteriophage”). Hereinafter, the difference between the present embodiment and the first embodiment will be mainly described.
　第２のバクテリオファージは、特に限定されないが、好ましくは、その宿主域が第１のバクテリオファージと異なる。さらに好ましくは、第２のバクテリオファージとして、第１のバクテリオファージによる溶菌活性が比較的低い青枯病菌の株に、第１のバクテリオファージによる溶菌活性よりも高い抗菌活性を示すものが挙げられる。好適には、第２のバクテリオファージは、第１のバクテリオファージが感染しない青枯病菌の株に感染する。また、第２のバクテリオファージは、その感染サイクルが、第１のバクテリオファージの感染サイクルと相違するものでもよい。 The second bacteriophage is not particularly limited, but preferably its host range is different from the first bacteriophage. More preferably, as the second bacteriophage, a strain of bacterial wilt that has a relatively low lytic activity by the first bacteriophage exhibits an antibacterial activity higher than that by the first bacteriophage. Preferably, the second bacteriophage is infected with a strain of bacterial wilt that is not infected by the first bacteriophage. The second bacteriophage may have a different infection cycle from the infection cycle of the first bacteriophage.
　具体的には、第２のバクテリオファージは、例えば、独立行政法人製品評価技術基盤機構特許微生物寄託センターに受託番号：ＮＩＴＥ　ＢＰ－１０８５として受託されたφＲＳＭ１、同じく受託番号：ＮＩＴＥ　ＢＰ－１０８６として受託されたφＲＳＭ３、特開２００７－２５２３５１号公報に開示されたＲＳＡ１、Ｔ７型のバクテリオファージであるＲＳＢ１、及びＲＳＬ２等である。なお、本実施の形態に係る青枯病防除剤は、第２のバクテリオファージとして、１種類のバクテリオファージに限らず、複数種類のバクテリオファージを含んでもよい。 Specifically, the second bacteriophage is, for example, φRSM1, which has been entrusted to the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation as NITE BP-1085, and the same as NITE BP-1086. ΦRSM3, RSA1 disclosed in Japanese Patent Application Laid-Open No. 2007-252351, RSB1, which is a T7 type bacteriophage, and RSL2. The bacterial wilt control agent according to the present embodiment is not limited to one type of bacteriophage as the second bacteriophage, and may include a plurality of types of bacteriophages.
　例えば、第１のバクテリオファージとしてのＲＳＦ１と、第２のバクテリオファージとしてのＲＳＢ１とを含む青枯病防除剤は、バクテリオファージとしてＲＳＦ１のみを含む青枯病防除剤と同様に、青枯病を防除できる。ＲＳＦ１とＲＳＢ１とを含む青枯病防除剤は、ＲＳＦ１の宿主域に含まれる青枯病菌及びＲＳＢ１の宿主域に含まれる青枯病菌のどちらにも有効である。 For example, a bacterial wilt control agent containing RSF1 as the first bacteriophage and RSB1 as the second bacteriophage has an effect against bacterial wilt like the bacterial wilt control agent containing only RSF1 as the bacteriophage. Can be controlled. The bacterial wilt control agent containing RSF1 and RSB1 is effective for both bacterial wilt fungi contained in the host range of RSF1 and bacterial wilt fungus contained in the host range of RSB1.
　本実施の形態に係る青枯病防除剤における第１のバクテリオファージと第２のバクテリオファージとの配合比は、特に限定されず、任意に設定すればよい。例えば、青枯病防除剤は、滅菌水に懸濁した状態で第１のバクテリオファージを、１０３～１０１４ｐｆｕ／ｍＬ、１０４～１０１２ｐｆｕ／ｍＬ又は１０３～１０８ｐｆｕ／ｍＬで含み、第２のバクテリオファージを１０３～１０１４ｐｆｕ／ｍＬ、１０４～１０１２ｐｆｕ／ｍＬ又は１０３～１０８ｐｆｕ／ｍＬで含む。 The blending ratio of the first bacteriophage and the second bacteriophage in the bacterial wilt control agent according to the present embodiment is not particularly limited and may be set arbitrarily. For example, the bacterial wilt control agent is obtained by suspending the first bacteriophage in a state of 10 3 to 10 14 pfu / mL, 10 4 to 10 12 pfu / mL, or 10 3 to 10 8 pfu / mL in a state of being suspended in sterilized water. And a second bacteriophage at 10 3 to 10 14 pfu / mL, 10 4 to 10 12 pfu / mL or 10 3 to 10 8 pfu / mL.
　以上詳細に説明したように、本実施の形態に係る青枯病防除剤は、青枯病菌に感染する複数種のバクテリオファージを含む。宿主域が異なるバクテリオファージを用いることで、さらに幅広い青枯病菌を溶菌させることができる。また、感染サイクルが異なるバクテリオファージを用いることで、多様な溶菌活性特性を得ることができる。 As described in detail above, the bacterial wilt control agent according to the present embodiment includes a plurality of types of bacteriophages that are infected with bacterial wilt. By using bacteriophages with different host ranges, a wider variety of bacterial wilt can be lysed. Moreover, various lytic activity characteristics can be obtained by using bacteriophages with different infection cycles.
　以下の実施例により、本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The following examples further illustrate the present invention, but the present invention is not limited to the examples.
　（実施例１：ＲＳＦ型ジャンボファージの単離及び精製）
　試験に用いた青枯病菌株は、０．１％（Ｗ／Ｖ）カザミノ酸、１．０％（Ｗ／Ｖ）ペプトン及び０．５％（Ｗ／Ｖ）グルコースを含有するＣＰＧ培地において、２８℃で振盪培養した（２００～３００ｒｐｍ）。 (Example 1: Isolation and purification of RSF type jumbo phage)
The bacterial wilt strain used for the test was a CPG medium containing 0.1% (W / V) casamino acid, 1.0% (W / V) peptone and 0.5% (W / V) glucose. The culture was shaken at 28 ° C. (200 to 300 rpm).
　広島県世羅郡世羅町で採取した土壌からバクテリオファージを、次のように単離した。１ｇの土壌を２ｍＬの滅菌水に懸濁して調製した試料を、室温で１時間、激しく振った。次に、膜孔の径が０．４５μｍの膜フィルター（Ｓｔｅｒａｄｉｓｃ、クラボウ社製）に試料を通し、１００μＬをプラークアッセイに用いた。プラークアッセイでは、青枯病菌株ＭＡＦＦ１０６６０３を宿主として、１．５％寒天を含むＣＰＧプレート上に重層した０．４５％の軟寒天培地にプラークを形成させた。 Bacteriophage was isolated from soil collected in Sera-cho, Sera-gun, Hiroshima as follows. A sample prepared by suspending 1 g of soil in 2 mL of sterile water was shaken vigorously at room temperature for 1 hour. Next, the sample was passed through a membrane filter having a pore diameter of 0.45 μm (Steradisc, manufactured by Kurabo Industries), and 100 μL was used for plaque assay. In the plaque assay, plaques were formed on a 0.45% soft agar medium overlaid on a CPG plate containing 1.5% agar using bacterial wilt strain MAFF106603 as a host.
　得られたバクテリオファージ（以下、「ＲＳＦ１」とする）を、ＭＡＦＦ１０６６０３を宿主として増殖させた。ＭＡＦＦ１０６６０３の培地のＯＤ６００が０．５に達してから、ＲＳＦ１をＭＯＩ（ｍｕｌｔｉｐｌｉｃｉｔｙ　ｏｆ　ｉｎｆｅｃｔｉｏｎ）＝０．０１～０．１で培地に加えた。１２～２４時間の培養後、ｈｉｍａｃ　ＣＲ２Ｅ遠心機（日立製作所製）のＲ１２Ａ２ローターを用いて、４℃で１５分間、８，０００×ｇで遠心することで細胞を除去した。膜孔の径が０．４５μｍの膜フィルターで上清を濾過し、さらに沈殿物をＳＭバッファー（５０ｍｍｏｌ／Ｌ　Ｔｒｉｓ－ＨＣｌ（ｐＨ　７．５）、１００ｍｍｏｌ／Ｌ　ＮａＣｌ、１０ｍｍｏｌ／Ｌ　ＭｇＳＯ４及び０．０１％ゼラチン）に溶解した。 The obtained bacteriophage (hereinafter referred to as “RSF1”) was grown using MAFF106603 as a host. After OD600 of the medium of MAFF106603 reached 0.5, RSF1 was added to the medium at MOI (multiplicity of effect) = 0.01-0.1. After culturing for 12 to 24 hours, cells were removed by centrifugation at 8,000 × g for 15 minutes at 4 ° C. using an R12A2 rotor of a himac CR2E centrifuge (manufactured by Hitachi, Ltd.). The supernatant was filtered with a membrane filter having a membrane pore diameter of 0.45 μm, and the precipitate was further filtered with SM buffer (50 mmol / L Tris-HCl (pH 7.5), 100 mmol / L NaCl, 10 mmol / L MgSO 4 and 0. (01% gelatin).
　ＲＳＦ１をさらに精製するために、ＲＳＦ１懸濁液をＣｓＣｌ（９．４ｇ／２０ｍＬ）に混合し、ＣＰ１００β超遠心機（日立製作所製）のＰ２８Ｓローターを用いて、１８時間、１４５，０００×ｇで超遠心した。精製されたＲＳＦ１を使用時まで４℃で保存した。 To further purify RSF1, the RSF1 suspension was mixed with CsCl (9.4 g / 20 mL) and 18 hours at 145,000 × g using the P28S rotor of a CP100β ultracentrifuge (Hitachi). Ultracentrifuged. Purified RSF1 was stored at 4 ° C. until use.
　（実施例２：ＲＳＦ１のファージ粒子の構造）
　ＲＳＦ１のファージ粒子（１０１２ｐｆｕ／ｍＬ）をリンタングステン酸でネガティブ染色し、電子顕微鏡（Ｈ６００Ａ、日立製作所製）で観察した。 (Example 2: Structure of RSF1 phage particle)
RSF1 phage particles (10 12 pfu / mL) were negatively stained with phosphotungstic acid and observed with an electron microscope (H600A, manufactured by Hitachi, Ltd.).
　（結果）
　図１に示すように、ＲＳＦ１のファージ粒子は、頭部が約１１５ｎｍの正二十面体、尾部の長さが１８０ｎｍ、尾部の幅が２５ｎｍのｍｙｏｖｉｒｕｓ型であった。なお、図１中のバーは１００ｎｍの長さを示す。 (result)
As shown in FIG. 1, the RSF1 phage particle was a myovirus type with an icosahedral head of about 115 nm, a tail length of 180 nm, and a tail width of 25 nm. The bar in FIG. 1 indicates a length of 100 nm.
　（実施例３：ＲＳＦ１のゲノムのサイズの決定）
　フェノール抽出によってファージ粒子からゲノムＤＮＡを単離した。ゲノムのサイズを決めるため、精製したファージ粒子を０．５％低融点アガロース（ＩｎＣｅｒｔ（商標）アガロース、ＦＭＣ社製）に包埋した。次に、１ｍｇ／ｍＬのプロテアーゼＫ（メルク社製）及び１％（Ｗ／Ｖ）のＳａｒｋｏｓｙｌで処理し、核酸に対してＣＨＥＦ　ＭＡＰＰＥＲ（商標）電気泳動装置（Ｂｉｏ－Ｒａｄ社製）でパルスフィールドゲル電気泳動法を実施した。 (Example 3: Determination of genome size of RSF1)
Genomic DNA was isolated from phage particles by phenol extraction. To determine the size of the genome, purified phage particles were embedded in 0.5% low melting point agarose (InCert ™ agarose, manufactured by FMC). Next, it was treated with 1 mg / mL protease K (manufactured by Merck) and 1% (W / V) Sarkosyl, and the nucleic acid was subjected to pulse field using a CHEF MAPPER ™ electrophoresis apparatus (manufactured by Bio-Rad). Gel electrophoresis was performed.
　（結果）
　図２は、パルスフィールドゲル電気泳動法で得られたバンドを示す。レーン１はサイズマーカーであるラムダラダーのバンドを示す。レーン２に示すＲＳＦ１のゲノムのサイズは、約２３０ｋｂｐであった。 (result)
FIG. 2 shows bands obtained by pulse field gel electrophoresis. Lane 1 shows a lambda ladder band which is a size marker. The genome size of RSF1 shown in lane 2 was about 230 kbp.
　（実施例４：ＲＳＦ１のゲノム解析）
　ＲＳＦ１のゲノムＤＮＡのショットガン配列決定をＧＳ　Ｊｕｎｉｏｒ　Ｓｅｑｕｅｎｃｅ　Ｓｙｓｔｅｍ（ロシュ社製）で行った。決定した塩基配列のアセンブリをＧＳ　Ｄｅ　Ｎｏｖｏ　Ａｓｓｅｍｂｌｅｒ　ｖ２．６で行った。解析された塩基配列は、２２２，８８８ｂｐであった。「ＡＴＧ」で始まる１５０ｂｐより大きいオープンリーディングフレーム（ＯＲＦ）をＧｌｉｍｍｅｒ　ｖ３．０２で同定した。配列データベースに対してＢＬＡＳＴＰ／ＲＰＳ－ＢＬＡＳＴでホモロジー検索を行った。ホモロジー検索では、著しい類似性のカットオフとして、Ｅ－ｖａｌｕｅが１ｅ－５未満とした。なお、配列データベースは、ＫＥＧＧ　ＧＥＮＥＳ、ＮＣＢＩ／Ｃｄｄ　ｓｅｑｕｅｎｃｅ　ｄｏｍａｉｎ　ｄａｔａｂａｓｅ（ｖｅｒｓｉｏｎ　３．１２）、ＵｎｉＰｒｏｔ　ｓｅｑｕｅｎｃｅ　ｄａｔａｂａｓｅ（Ｒｅｌｅａｓｅ　２０１４＿０８）及びＮＣＢＩ　ＲｅｆＳｅｑ　ｃｏｍｐｌｅｔｅ　ｖｉｒａｌ　ｇｅｎｏｍｅ　ｓｅｃｔｉｏｎ　ｄａｔａｂａｓｅ（Ｒｅｌｅａｓｅ　６７、２０１４年９月８日）である。ｔＲＮＡ遺伝子は、ｔＲＮＡＳｃａｎ－ＳＥ　１．４（ｏｐｔｉｏｎ；－Ｂ　ｆｏｒ　ｂａｃｔｅｒｉａｌ　ｔＲＮＡｓ）を用いて同定した。環状ゲノム地図は、ＣＧＶｉｅｗで描いた。 (Example 4: Genomic analysis of RSF1)
Shotgun sequencing of RSF1 genomic DNA was performed on a GS Junior Sequence System (Roche). Assembly of the determined base sequence was performed with GS De Novo Assembler v2.6. The analyzed base sequence was 222,888 bp. An open reading frame (ORF) greater than 150 bp beginning with “ATG” was identified with Glimmer v3.02. A homology search was performed on the sequence database using BLASTP / RPS-BLAST. In the homology search, E-value was less than 1e-5 as a significant similarity cut-off. The sequence databases are KEGG GENES, NCBI / Cdd sequence domain database (version 3.12), UniProt sequence database (Release 2014_08), NCBI Refseq complete 20 . The tRNA genes were identified using tRNAScan-SE 1.4 (option; -B for bacterial tRNAs). The circular genome map was drawn with CGView.
　（結果）
　ＲＳＦ１の環状ゲノム地図を図３に示す。ゲノムは環状に重複した２２２，８８８ｂｐの二重鎖ＤＮＡであった。ゲノムには、計２３０個の遺伝子がコードされていた。２３０個の遺伝子のうち、５５個が時計回りにコードされていて、残りが反時計回りにコードされていた。配列データベースで生物学的に特徴づけられていたタンパク質との類似性に基づいて、２７個のＯＲＦのアノテーションを決定することができた。 (result)
A circular genome map of RSF1 is shown in FIG. The genome was a 222,888 bp double-stranded DNA with circular overlap. A total of 230 genes were encoded in the genome. Of the 230 genes, 55 were encoded clockwise and the rest were encoded counterclockwise. Based on the similarity to proteins that were biologically characterized in the sequence database, 27 ORF annotations could be determined.
　ビリオン関連ＲＮＡポリメラーゼのβサブユニット（ＲｐｏＢ）のＮ領域及びＣ領域は、それぞれＯＲＦ４０及びＯＲＦ５１にコードされていた。ビリオン関連ＲＮＡポリメラーゼのβ’サブユニット（ＲｐｏＣ）のＮ領域及びＣ領域は、それぞれＯＲＦ４１及びＯＲＦ１９９にコードされていた。一方、初期発現ＲＮＡポリメラーゼのβサブユニット（ＲｐｏＢ）のＮ領域及びＣ領域は、それぞれＯＲＦ１２２及びＯＲＦ２１５にコードされていた。初期発現ＲＮＡポリメラーゼのβ’サブユニット（ＲｐｏＣ）のＮ領域及びＣ領域は、それぞれＯＲＦ２２７及びＯＲＦ２１４にコードされていた。 The N region and C region of the β subunit (RpoB) of virion-related RNA polymerase were encoded by ORF40 and ORF51, respectively. The N region and C region of the β 'subunit (RpoC) of virion-related RNA polymerase were encoded by ORF41 and ORF199, respectively. On the other hand, the N region and C region of the β subunit (RpoB) of the initially expressed RNA polymerase were encoded by ORF122 and ORF215, respectively. The N region and C region of the β ′ subunit (RpoC) of the early expressed RNA polymerase were encoded by ORF227 and ORF214, respectively.
　また、ＲＳＦ１のゲノムにおいて溶菌酵素であるＬｙｓＭ様ムレイン溶解酵素（キチナーゼ様）は、ＯＲＦ４２にコードされていた。また、溶菌酵素であるＳＬＴ糖転移酵素は、ＯＲＦ５５にコードされていた。 In addition, LysM-like murein lytic enzyme (chitinase-like), which is a lytic enzyme in RSF1 genome, was encoded by ORF42. In addition, SLT glycosyltransferase, which is a lytic enzyme, was encoded by ORF55.
　ＲＳＦ１のゲノムにおいてＤＮＡ複製に関連するタンパク質の遺伝子のホモログであるＯＲＦとしては、ＯＲＦ５７（ＲＮａｓｅ　Ｈ）、ＯＲＦ６３（ＳｂｃＣ－ＡＴＰａｓｅ）、ＯＲＦ１０５（ＤＮＡリガーゼ）及びＯＲＦ１２６（ＤｎａＢヘリカーゼ）が挙げられる。また、ＯＲＦ６８はＧＩＹ－ＹＩＧ型ヌクレアーゼに類似性が高い。 ORFs that are homologues of proteins related to DNA replication in the genome of RSF1 include ORF57 (RNase H), ORF63 (SbcC-ATPase), ORF105 (DNA ligase) and ORF126 (DnaB helicase). ORF68 is highly similar to GIY-YIG type nuclease.
　ＲＳＦ１のゲノムに含まれるヌクレオチドの代謝に関連する酵素のホモログとしては、ＯＲＦ１９０（チミジル酸キナーゼ）、ＯＲＦ１６４（チミジル酸シンターゼ）、ＯＲＦ７７及びＯＲＦ７８（ジヒドロ葉酸レダクターゼ）、ＯＲＦ１１８（リボヌクレオチドレダクターゼ　αサブユニット）、ＯＲＦ１１７（リボヌクレオチドレダクターゼ　βサブユニット）及びＯＲＦ１１９（嫌気性リボヌクレオチド二リン酸レダクターゼ）が挙げられる。 As homologues of enzymes related to the metabolism of nucleotides contained in the genome of RSF1, ORF190 (thymidylate kinase), ORF164 (thymidylate synthase), ORF77 and ORF78 (dihydrofolate reductase), ORF118 (ribonucleotide reductase α subunit) ORF117 (ribonucleotide reductase β subunit) and ORF119 (anaerobic ribonucleotide diphosphate reductase).
　上記の他、宿主又はファージ相互作用関連のタンパク質及びファージ粒子を構成するタンパク質をコードする遺伝子に相同性を有するＯＲＦが同定された。 In addition to the above, ORFs having homology to the host or phage interaction-related protein and the gene encoding the protein constituting the phage particle were identified.
　（実施例５：ｎａｎｏＬＣ－ＥＩＳ　ＭＳ／ＭＳによる構造タンパク質の同定）
　精製したファージ粒子について公知の方法でＳＤＳ－ＰＡＧＥ（１０～１２％（Ｗ／Ｖ）ポリアクリルアミド）を行った。タンパク質のバンドは、クマシーブリリアントブルーでゲルを染色することで可視化し、ゲルから切り出して、ジチオスレイトールで還元し、ヨードアセトアミドでアルキル化した後、トリプシンで断片化した。トリプシンペプチドをｓｈｏｒｔ　ＯＤＳ　ｃｏｌｕｍｎ（ＰｅｐＭａｐ　１００；５μｍ　Ｃ１８、５ｍｍ×３００μｍ　ＩＤ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用いてトラップし、ＯＤＳ　ｃｏｌｕｍｎ（Ｎａｎｏ　ＨＰＬＣ　Ｃａｐｉｌｌａｒｙ　Ｃｏｌｕｍｎ、３μｍ　Ｃ１８、１２０ｍｍ×７５μｍ　ＩＤ、日京テクノス社製）を用いたｎａｎｏ－ｌｉｑｕｉｄ　ｃｈｒｏｍａｔｏｇｒａｐｈｙ（ｎａｎｏＬＣ）で分離した。ｎａｎｏＬＣには、Ｕｌｔｉｍａｔｅ（商標）　３０００　ＲＳＬＣ　ｎａｎｏ　ｓｙｓｔｅｍ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を使用した。 (Example 5: Identification of structural protein by nanoLC-EIS MS / MS)
The purified phage particles were subjected to SDS-PAGE (10-12% (W / V) polyacrylamide) by a known method. Protein bands were visualized by staining the gel with Coomassie Brilliant Blue, excised from the gel, reduced with dithiothreitol, alkylated with iodoacetamide, and then fragmented with trypsin. The trypsin peptide was trapped using a short ODS column (PepMap 100; 5 μm C18, 5 mm × 300 μm ID, manufactured by Thermo Fisher Scientific), and ODS column (Nano HPLC Capillary Column, 3 × 75 C18, 3 μm C18mm The product was separated using a nano-liquid chromatography (nanoLC). For nanoLC, Ultimate ™ 3000 RSLC nano system (manufactured by Thermo Fisher Scientific) was used.
　分離における移動相は、溶媒Ａ（０．１％ギ酸）と溶媒Ｂ（アセトニトリル中の０．１％ギ酸）とした。流速３０μＬ／分で３分間、０．１％ＴＦＡでトラップカラムにトリプシンペプチドをロードした後、濃縮されたトリプシンペプチドをトラップカラムから溶出し、一連のアイソクラティック法と直線的濃度勾配法（０～３分が溶媒Ａ、３～３５分が０～３５％（ｖ／ｖ）の溶媒Ｂ、そして１０分で９０％溶媒Ｂに増加して溶媒Ａで１５分の再平衡化）を用いて分離カラムで分離した。分離カラムからの溶出液をｎａｎｏＥＳＩソースに継続的に供給し、ＭＳ及びＭＳ／ＭＳ（ＬＴＱ　Ｏｒｂｉｔｒａｐ　ＸＬ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）で解析した。ＭＳスペクトル及びＭＳ／ＭＳスペクトルは、それぞれＯｒｂｉｔｒａｐ（マスレンジ：ｍ／ｚ　３００～１５００）及びＩｏｎｔｒａｐ（ＣＩＤを用いた上位５ピークのスキャン依存データ）を用いて陽イオンモードで取得した。 The mobile phases in the separation were solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile). After loading trypsin peptide onto the trap column with 0.1% TFA for 3 minutes at a flow rate of 30 μL / min, the concentrated tryptic peptide was eluted from the trap column and a series of isocratic and linear gradient (0 ~ 3 minutes solvent A, 3 ~ 35 minutes 0-35% (v / v) solvent B, and 10 minutes increase to 90% solvent B and 15 minutes re-equilibration with solvent A) Separation with a separation column. The eluate from the separation column was continuously supplied to the nanoESI source and analyzed with MS and MS / MS (LTQ Orbitrap XL, manufactured by Thermo Fisher Scientific). MS spectra and MS / MS spectra were obtained in positive ion mode using Orbitrap (mass range: m / z 300-1500) and Iontrap (scanning dependent data of top 5 peaks using CID), respectively.
　キャピラリソースの電圧は１．５ｋＶに設定し、トランスファーキャピラリの温度は２００℃に維持した。キャピラリ電圧及びチューブレンズ電圧は、それぞれ２０Ｖ及び８０Ｖとした。ＲＳＦ１のＯＲＦにコードされるトリプシンペプチドへのＭＳ／ＭＳデータの割り当ては、Ｘｃａｌｉｂｕｒプログラム（ｖｅｒ２．０、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用いて行なった。 The capillary source voltage was set to 1.5 kV and the transfer capillary temperature was maintained at 200 ° C. The capillary voltage and tube lens voltage were 20 V and 80 V, respectively. Assignment of MS / MS data to the tryptic peptide encoded by the ORF of RSF1 was performed using the Xcalibur program (ver2.0, manufactured by Thermo Fisher Scientific).
　（結果）
　図４はＲＳＦ１の構造タンパク質を分離したＳＤＳ－ＰＡＧＥのバンドを示す。ＲＳＦ１のファージ粒子を構成するタンパク質に、ＯＲＦ４０、ＯＲＦ５１、ＯＲＦ４１及びＯＲＦ１９９由来のタンパク質が含まれていた。 (result)
FIG. 4 shows the SDS-PAGE band from which the structural protein of RSF1 was separated. Proteins constituting RSF1 phage particles included proteins derived from ORF40, ORF51, ORF41, and ORF199.
　比較のため、上記非特許文献１に開示されたジャンボファージＪ６（以下では、「ＲＳＬ２」とする）の構造タンパク質を同様に同定した。ＲＳＬ２はゲノムのサイズが約２２０ｋｂｐで、ＲＳＦ１と同様にファージ粒子がｍｙｏｖｉｒｕｓ型である。図５に示すように、ＲＳＬ２のファージ粒子を構成する構造タンパク質には、ビリオン関連ＲＮＡポリメラーゼのβサブユニットのＮ領域及びＣ領域をそれぞれコードするＯＲＦ３７及びＯＲＦ４８由来のタンパク質と、β’サブユニットのＣ領域をコードするＯＲＦ１９２由来のタンパク質が検出されたが、β’サブユニットのＮ領域をコードするＯＲＦ３８由来のタンパク質は検出されなかった。 For comparison, the structural protein of jumbo phage J6 (hereinafter referred to as “RSL2”) disclosed in Non-Patent Document 1 was similarly identified. RSL2 has a genome size of about 220 kbp, and the phage particle is a myovirus type like RSF1. As shown in FIG. 5, the structural proteins constituting RSL2 phage particles include ORF37 and ORF48-derived proteins encoding the N and C regions of the β subunit of virion-related RNA polymerase, respectively, and β ′ subunit A protein derived from ORF192 encoding the C region was detected, but a protein derived from ORF38 encoding the N region of the β ′ subunit was not detected.
　（実施例６：ＲＳＦ１の宿主域の検討）
　種々の青枯病菌株を用いた上記のプラークアッセイによって、ＲＳＦ１の宿主域を検討した。さらに、比較のため、ＲＳＬ２の宿主域も同様に検討した。 (Example 6: Examination of host range of RSF1)
The host range of RSF1 was examined by the above plaque assay using various bacterial wilt strains. Furthermore, the host range of RSL2 was also examined for comparison.
　（結果）
　表１は、ＲＳＬ２及びＲＳＦ１の宿主域を示す。ＲＳＬ２又はＲＳＦ１に対する感受性に関しては、「＋」は感受性があることを示し、「－」は感受性がない（抵抗性である）ことを示す。ＲＳＦ１は種々の植物を宿主植物とする１９種類の菌株に感染する。ＲＳＦ１が感染する菌株には、ＲＳＬ２が感染しないＰｓ６５、ＭＡＦＦ２１１５１４、ＭＡＦＦ３０１４８５及びＭＡＦＦ３０１５５８が含まれており、ＲＳＦ１は、ＲＳＬ２と比較して、日本由来の青枯病菌株に対して広い宿主域を示した。また、ＲＳＦ１は青枯病菌のみに感染し、腸内細菌、Ｐｓｅｕｄｏｍｏｎａｓ、Ｒｈｉｚｏｂｉｕｍ及びグラム陽性菌などには感染しない。 (result)
Table 1 shows the host range of RSL2 and RSF1. Regarding sensitivity to RSL2 or RSF1, “+” indicates sensitivity and “−” indicates insensitivity (resistance). RSF1 infects 19 strains of various plants as host plants. The strains infected with RSF1 include Ps65, MAFF21114, MAFF301485 and MAFF301558, which are not infected with RSL2, and RSF1 showed a broad host range for bacterial wilt strains derived from Japan compared to RSL2. . RSF1 only infects bacterial wilt and does not infect enterobacteria, Pseudomonas, Rhizobium, Gram-positive bacteria, and the like.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　（実施例７：ＲＳＦ１の感染サイクルの評価）
　ワンステップ増殖法で感染サイクルを評価した。培養によりＯＤ６００が０．１に達したＭＡＦＦ７３０１３８株を遠心（６０００×ｇ）で回収し、最終培養液量が１０ｍＬとなるようにＣＰＧ培地に懸濁した（およそ１×１０８ｃｆｕ／ｍＬ）。ＲＳＦ１をＭＯＩ＝０．１となるように加えて、２８℃で１０分間吸着させた。遠心後、当初の量のＣＰＧに試料を再懸濁し、最終液量が１０ｍＬになるように希釈系列を調製した。細胞を２８℃でインキュベーションした。試料を３０分ごとに採取し、タイターをプラークアッセイで決定した。 (Example 7: Evaluation of infection cycle of RSF1)
The infection cycle was evaluated by a one-step growth method. The MAFF730138 strain having an OD600 of 0.1 by culture was collected by centrifugation (6000 × g) and suspended in CPG medium so that the final culture volume was 10 mL (approximately 1 × 10 8 cfu / mL). RSF1 was added so that MOI = 0.1, and adsorbed at 28 ° C. for 10 minutes. After centrifugation, the sample was resuspended in the initial amount of CPG, and a dilution series was prepared so that the final liquid volume was 10 mL. Cells were incubated at 28 ° C. Samples were taken every 30 minutes and titers were determined by plaque assay.
　（結果）
　細胞あたりのＲＳＦ１の数の経時変化を図６に示す。ＲＳＦ１は、ＭＡＦＦ７３０１３８株を宿主とした場合、潜伏期が９０分、かつ４時間の感染サイクルで、バーストサイズが約８０ｐｆｕ／細胞であった。なお、ＲＳＬ２の感染サイクルを同様に評価すると、潜伏期が１５０分、かつ４．５時間の感染サイクルで、バーストサイズが約４０～５０ｐｆｕ／細胞であった。ＲＳＬ２は、ファージ粒子にビリオン関連ＲＮＡポリメラーゼのβ’サブユニットの一部を欠くため、初期の発現に宿主のＲＮＡポリメラーゼに依存するのに対し、ＲＳＦ１は、ファージ粒子にビリオン関連ＲＮＡポリメラーゼのβサブユニット及びβ’サブユニットをフルセットで有するため、潜伏期及び感染サイクルが短いと考えられる。これにより、ＲＳＦ１の感染効率が、ＲＳＬ２よりも高められている。 (result)
The time course of the number of RSF1 per cell is shown in FIG. When the MAFF730138 strain was used as a host, RSF1 had an incubation period of 90 minutes and an infection cycle of 4 hours, and the burst size was about 80 pfu / cell. When the infection cycle of RSL2 was evaluated in the same manner, the burst size was about 40-50 pfu / cell with an incubation period of 150 minutes and 4.5 hours of incubation period. RSF2 relies on the host RNA polymerase for early expression because RSL2 lacks part of the β ′ subunit of the virion-related RNA polymerase in the phage particle, whereas RSF1 relies on the β-subunit of the virion-related RNA polymerase in the phage particle. Because it has a full set of units and β ′ subunits, the incubation period and infection cycle are considered short. Thereby, the infection efficiency of RSF1 is higher than RSL2.
　（実施例８：トマトにおけるＲＳＦ１の青枯病防除効果の評価）
　青枯病菌株ＭＡＦＦ２１１５１４を、２８℃で１～２日、ＣＰＧ培地で培養した。遠心後、細胞を１．５×１０９細胞／ｍＬ（ＯＤ６００＝１．０）の濃度で滅菌水に懸濁した。５ｍＬの細胞懸濁液を、断根したトマト苗（Ｓｏｌａｎｕｍ　ｌｙｃｏｐｅｒｓｉｃｕｍ　Ｌ．、品種「大型福寿」）のポット内の土壌に投与した（対照区）。なお、トマト苗として、葉が４～６枚の１ヶ月苗を用いた。土壌１ｇあたりの細胞の濃度は、約１×１０６ｃｆｕである。ファージ処理区には、細胞懸濁液を投与する１日前に、５ｍＬのＲＳＦ１懸濁液（１．５×１０１０ｐｆｕ）を苗根部に添加した。 (Example 8: Evaluation of bacterial wilt control effect of RSF1 in tomato)
The bacterial wilt strain MAFF 211514 was cultured in CPG medium at 28 ° C. for 1-2 days. After centrifugation, the cells were suspended in sterile water at a concentration of 1.5 × 10 9 cells / mL (OD600 = 1.0). 5 mL of the cell suspension was administered to the soil in the pot of the rooted tomato seedling (Solanum lycopersicum L., variety “Large Fuju”) (control group). As tomato seedlings, 1-month seedlings with 4 to 6 leaves were used. The concentration of cells per gram of soil is about 1 × 10 6 cfu. In the phage-treated group, 5 mL of RSF1 suspension (1.5 × 10 10 pfu) was added to the seedling root part one day before administration of the cell suspension.
　（結果）
　図７（Ａ）及び（Ｂ）は、それぞれ対照区及びファージ処理区のトマト苗に現れた病徴を示す。病徴指数「０」は変化なし、「１」は子葉から上に向かい第１葉が萎凋、「２」は第２葉が萎凋、「３」は第３葉が萎凋、「４」は第４葉が萎凋、「５」は枯死、を示す。図７（Ａ）に示すように、対照区のトマト苗には、細胞懸濁液の投与から約１週間後に顕著な青枯病の病徴が出現した。約２週間後には対照区のトマト苗の８０％が枯死した。一方、図７（Ｂ）に示すように、ファージ処理区では、３週間後でもトマト苗に変化がなかった。その後、ファージ処理区のトマト苗には、少し萎えた第１葉が若干観察されたがこれは青枯病の病徴とは異なっていた。ファージ処理区のトマト苗は、１ヶ月後でも青枯病の発症はなかった。また、潜在的に発生するＲＳＦ１に対する耐性菌は、病原性を喪失していると考えられる。 (result)
FIGS. 7 (A) and (B) show the symptom appearing in the tomato seedlings in the control group and the phage-treated group, respectively. Symptom index “0” is unchanged, “1” is upward from the cotyledon, the first leaf is wilt, “2” is wilt the second leaf, “3” is wilt the third leaf, “4” is the first Four leaves indicate wilting and “5” indicates death. As shown in FIG. 7 (A), a remarkable symptom of bacterial wilt appeared about 1 week after administration of the cell suspension in the tomato seedlings in the control group. After about 2 weeks, 80% of the tomato seedlings in the control group died. On the other hand, as shown in FIG. 7 (B), the tomato seedlings did not change even after 3 weeks in the phage-treated group. Thereafter, the first leaves slightly withered were observed in the tomato seedlings in the phage-treated group, which was different from the symptoms of bacterial wilt. The tomato seedlings in the phage-treated area did not develop bacterial wilt even after one month. Moreover, it is thought that the resistant microbe to RSF1 which generate | occur | produces has lost pathogenicity.
　本発明は、本発明の広義の精神と範囲を逸脱することなく、様々な実施の形態及び変形が可能とされるものである。また、上述した実施の形態は、本発明を説明するためのものであり、本発明の範囲を限定するものではない。すなわち、本発明の範囲は、実施の形態ではなく、特許請求の範囲によって示される。そして、特許請求の範囲内及びそれと同等な発明の意義の範囲内で施される様々な変形が、本発明の範囲内とみなされる。 The present invention is capable of various embodiments and modifications without departing from the broad spirit and scope of the present invention. The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.
　本出願は、２０１５年１２月１７日に出願された日本国特許出願２０１５－２４５７７２号に基づく。本明細書中に、日本国特許出願２０１５－２４５７７２号の明細書、特許請求の範囲、図面全体を参照として取り込むものとする。 This application is based on Japanese Patent Application No. 2015-245772 filed on December 17, 2015. In this specification, the specification, claims, and entire drawings of Japanese Patent Application No. 2015-245772 are incorporated by reference.
　本発明は、青枯病の防除、拡大防止又は青枯病菌の駆除に好適である。 The present invention is suitable for controlling bacterial wilt, preventing spreading, or controlling bacterial wilt.

Claims (6)

  1. 　ゲノムのサイズが２００，０００ｂｐ以上であって、
    　ファージ粒子を構成するタンパク質に、ビリオン関連ＲＮＡポリメラーゼのβサブユニットとビリオン関連ＲＮＡポリメラーゼのβ’サブユニットとを含み、
    　Ｒａｌｓｔｏｎｉａ　ｓｏｌａｎａｃｅａｒｕｍに感染する、
    　バクテリオファージ。     The genome size is over 200,000 bp,
    The protein constituting the phage particle includes a β subunit of a virion-related RNA polymerase and a β ′ subunit of a virion-related RNA polymerase,
    Infects Ralstonia solanacearum,
    Bacteriophage.
  2. 　前記ゲノムには、
    　２種類以上の溶菌酵素がコードされている、
    　請求項１に記載のバクテリオファージ。     The genome includes
    Two or more lytic enzymes are encoded,
    The bacteriophage according to claim 1.
  3. 　独立行政法人製品評価技術基盤機構特許微生物寄託センターに２０１５年１２月１０日に受託された受託番号：ＮＩＴＥ　ＢＰ－０２１７６で示されるＲＳＦ１である、
    　請求項１又は２に記載のバクテリオファージ。     RSF1 indicated by NITE BP-02176, which was deposited on December 10, 2015 at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation for Product Evaluation Technology,
    The bacteriophage according to claim 1 or 2.
  4. 　請求項１から３のいずれか一項に記載のバクテリオファージを含む、
    　青枯病防除剤。     Comprising the bacteriophage according to any one of claims 1 to 3,
    A bacterial blight control agent.
  5. 　請求項１から３のいずれか一項に記載のバクテリオファージと、
    　前記バクテリオファージと異なり、かつＲａｌｓｔｏｎｉａ　ｓｏｌａｎａｃｅａｒｕｍに感染するバクテリオファージと、
    　を含む、青枯病防除剤。     Bacteriophage according to any one of claims 1 to 3,
    A bacteriophage that is different from the bacteriophage and infects Ralstonia solanacerum;
    A bacterial wilt control agent.
  6. 　請求項４又は５に記載の青枯病防除剤を、植物又は植物成長媒体に投与する投与ステップを含む、
    　青枯病防除方法。     An administration step of administering the bacterial wilt control agent according to claim 4 or 5 to a plant or a plant growth medium,
    How to control bacterial wilt.
PCT/JP2016/084291 2015-12-17 2016-11-18 Bacteriophage, bacterial wilt disease control agent, and method for controlling bacterial wilt disease WO2017104347A1 (en)

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