WO2017104347A1 - Bactériophage, agent de lutte contre la maladie du flétrissement bactérien, et méthode pour luuter contre la maladie du flétrissement bactérien - Google Patents

Bactériophage, agent de lutte contre la maladie du flétrissement bactérien, et méthode pour luuter contre la maladie du flétrissement bactérien 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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English (en)
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

L'invention concerne un bactériophage ayant une taille de génome de 200 000 bp ou plus, une protéine constituant les particules de phage du bactériophage contenant une sous-unité β d'ARN polymérase associée à un virion et une sous-unité β' d'ARN polymérase associée à un virion. Le bactériophage peut être transmis à Ralstonia solanacearum.
PCT/JP2016/084291 2015-12-17 2016-11-18 Bactériophage, agent de lutte contre la maladie du flétrissement bactérien, et méthode pour luuter contre la maladie du flétrissement bactérien WO2017104347A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108410825A (zh) * 2018-04-20 2018-08-17 南京农业大学 一种噬菌体鸡尾酒及其应用
WO2022024287A1 (fr) * 2020-07-30 2022-02-03 パネフリ工業株式会社 Bactériophage, agent de lutte contre la flétrissure bactérienne et procédé de lutte contre la flétrissure bactérienne
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CN115261338A (zh) * 2022-08-15 2022-11-01 福建省农业科学院植物保护研究所 一株具有防控烟草青枯病的裂解性噬菌体s5及应用
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