WO2024248028A1 - キサントモナス属細菌溶菌性バクテリオファージ - Google Patents

キサントモナス属細菌溶菌性バクテリオファージ Download PDF

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WO2024248028A1
WO2024248028A1 PCT/JP2024/019668 JP2024019668W WO2024248028A1 WO 2024248028 A1 WO2024248028 A1 WO 2024248028A1 JP 2024019668 W JP2024019668 W JP 2024019668W WO 2024248028 A1 WO2024248028 A1 WO 2024248028A1
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phage
bacteria
seq
plant
genomic dna
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French (fr)
Japanese (ja)
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慎一 吉田
暢彦 道順
英知 岩野
仁 工藤
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RAKUNO GAKUEN UNIVERSITY
Kaneka Corp
Mitsui and Co Ltd
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RAKUNO GAKUEN UNIVERSITY
Kaneka Corp
Mitsui and Co Ltd
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Priority to JP2025524126A priority Critical patent/JPWO2024248028A1/ja
Priority to EP24815509.5A priority patent/EP4722351A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to a lytic agent comprising a bacteriophage, a plant disease control composition containing the same, and a plant disease control method.
  • Bacteriophage (often abbreviated simply as "phage” in this specification) is a general term for viruses that infect only bacteria. Many phages, also called lytic phages, specifically adsorb to the target bacterium (host), inject their own DNA, and self-amplify using the translation mechanism of the bacterium. Furthermore, they disseminate the amplified phages by lysing the bacterium, and repeatedly infect new target bacteria (Non-Patent Document 1).
  • Non-Patent Document 2 a method that applies phages has attracted attention as a new control method
  • Patent documents 1 to 3 and non-patent document 2 report examples of phages that exhibit lytic activity against Xanthomonas bacteria. However, because the host range of phages is extremely narrow, it remains important to search for new phages and discover phages with higher lytic activity.
  • phages are viruses and natural products, so no reports of phages causing any chemical damage have been reported.
  • phages are viruses and natural products, so no reports of phages causing any chemical damage have been reported.
  • they because they have high specificity for the host, only specific genus or species of bacteria are targeted, and the impact on the balance of the microflora is extremely limited. They are also harmless to not only animals, including humans, but also plants, and are highly safe. Therefore, in order to suppress damage to agricultural crops caused by plant diseases caused by Xanthomonas bacteria, a novel phage that exhibits bacteriolytic activity against Xanthomonas bacteria is isolated.
  • the present invention aims to provide a composition containing the phage as an active ingredient, and to use the composition for disease control, pathogenic bacteria detection, etc.
  • the inventors isolated novel phages from natural wastewater and soil using a method for detecting lytic plaques formed on soft agar medium cultured with Xanthomonas bacteria, and analyzed their genome sequences. As a result, it was revealed that the phages possess novel genome DNA sequences with no related genome sequences known at all.
  • the present invention was completed based on the results of the above research and development. Specifically, it provides the following.
  • a lytic agent comprising a bacteriophage having a genomic DNA sequence containing any of the following base sequences (1a) to (1c): (1a) the base sequence shown in SEQ ID NO: 1, (1b) a base sequence in which one or more bases have been added, deleted, and/or substituted in the base sequence shown in SEQ ID NO: 1, or (1c) a base sequence having 95% or more sequence identity with the base sequence shown in SEQ ID NO: 1.
  • the lytic agent according to [1-A] which exhibits lytic activity against Xanthomonas bacteria.
  • [3-A] The lytic agent according to [2-A], wherein the Xanthomonas bacterium is at least one bacterium selected from the group consisting of Xanthomonas arboricola, Xanthomonas campestris, and Xanthomonas citri.
  • a lytic agent comprising a bacteriophage having a genomic DNA sequence having a total length of 40,000 bases or less, comprising any one of the following base sequences (2a) to (2c): (2a) the base sequence shown in SEQ ID NO: 2, (2b) the base sequence shown in SEQ ID NO: 2 in which one or more bases have been added, deleted, and/or substituted, or (2c) a base sequence having 90% or more sequence identity to the base sequence shown in SEQ ID NO: 2.
  • the lytic agent according to [1-B] which exhibits lytic activity against Xanthomonas bacteria.
  • a lytic agent comprising a bacteriophage having a genomic DNA sequence containing any one of the following base sequences (3a) to (3c): (3a) the base sequence shown in SEQ ID NO: 3, (3b) the base sequence shown in SEQ ID NO: 3 in which one or more bases have been added, deleted, and/or substituted, or (3c) a base sequence having 90% or more sequence identity to the base sequence shown in SEQ ID NO: 3.
  • a composition comprising, as an active ingredient, the lytic agent according to any one of [1-A] to [3-C].
  • [5] A plant disease control composition comprising the composition according to [4].
  • a method for identifying Xanthomonas bacteria comprising a culture step of culturing a test bacterium isolated from plant tissue infected with a plant disease to obtain a culture, a mixing step of mixing the culture with a lytic agent described in [2] or [3] to obtain a mixture, a mixture culture step of culturing the mixture under specified conditions, and a determination step of determining that the test bacterium is a Xanthomonas bacterium if the test bacterium has been lysed after the mixture culture step.
  • the mixture further contains a soft agar-containing liquid medium, and the mixture is cultured on a solid medium.
  • the bacteriolytic agent of the present invention and a composition containing it as an active ingredient can lyse specific target bacteria.
  • the plant disease control composition of the present invention can prevent and suppress diseases caused by specific target bacteria.
  • Figure 1 shows the lytic activity of the first bacteriophage obtained in Example 1.
  • Figure 1A shows the Xanthomonas bacteria shown in Table 2 spread on an agar plate, and the control Pseudomonas fluorescens, which were then cultured, and the first phage purified solution was dropped onto the center of the plate, followed by static culture.
  • Figure 1B shows the plate corresponding to Figure 1A, showing the bacteria shown in Table 2 spread on each plate. This shows the lytic activity of the first bacteriophage obtained in Example 1.
  • A is a diagram of Xanthomonas bacteria and control Pseudomonas fluorescens cultured on an agar plate, after which the first phage purified solution was dropped onto the center of the plate and allowed to stand for static culture.
  • B is a diagram of the plate corresponding to A, showing the bacteria shown in Table 2 spread on each plate. This shows the lytic activity of the second bacteriophage obtained in Example 2.
  • A is a diagram of an agar plate on which Xanthomonas bacteria shown in Table 4 and control Pseudomonas fluorescens were cultured, and then the second phage purified solution was dropped onto the center of the plate and allowed to stand for static culture.
  • B is a diagram of the plate corresponding to A, showing the bacteria shown in Table 4 spread on each plate. This shows the lytic activity of the second bacteriophage obtained in Example 2.
  • A is a diagram of an agar plate on which Xanthomonas bacteria shown in Table 4 and control Pseudomonas fluorescens were cultured, and then the second phage purified solution was dropped onto the center of the plate and allowed to stand for static culture.
  • B is a diagram of the plate corresponding to A, showing the bacteria shown in Table 4 spread on each plate. This shows the lytic activity of the third bacteriophage obtained in Example 3.
  • A is a diagram of an agar plate on which Xanthomonas bacteria shown in Table 5 and control Pseudomonas fluorescens were cultured, and then the third phage purified solution was dropped onto the center of the plate and allowed to stand for static culture.
  • B is a diagram of the plate corresponding to A, showing the bacteria shown in Table 5 spread on each plate. This figure shows the lytic activity of the combination of the first bacteriophage and other bacteriophages tested in Example 4.
  • A is a diagram of Xanthomonas bacteria spread on an agar plate, which was then cultured, and the purified phage solution was dropped onto the plate and allowed to stand.
  • FIG. 6A and B is a plate diagram corresponding to A, showing the type of phage contained in the purified phage solution dropped onto each plate.
  • the left two columns (1, 2, 5, 6, 9, and 10) show plates on which bacteria with ID MAFF No. 673005 were spread
  • the right two columns (3, 4, 7, 8, 11, and 12) show plates on which bacteria with ID MAFF No. 301352 were spread.
  • a indicates a first phage having a genomic DNA sequence of SEQ ID NO: 1
  • b indicates a phage having a genomic DNA sequence of SEQ ID NO: 4
  • c indicates a phage having a genomic DNA sequence of SEQ ID NO: 5
  • d indicates a phage having a genomic DNA sequence of SEQ ID NO: 6
  • e indicates a phage having a genomic DNA sequence of SEQ ID NO: 7
  • f indicates a second phage having a genomic DNA sequence of SEQ ID NO: 2
  • g indicates a third phage having a genomic DNA sequence of SEQ ID NO: 3
  • h indicates a phage having a genomic DNA sequence of SEQ ID NO: 8.
  • A is a diagram of Xanthomonas bacteria spread on an agar plate, which was then cultured, and the purified phage solution was dropped onto the plate and allowed to stand.
  • B is a plate diagram corresponding to A, showing the type of phage contained in the purified phage solution dropped onto each plate.
  • the two left columns (1, 2, 5, 6, 9, and 10) show plates on which bacteria with ID MAFF No.
  • a indicates the second phage having the genomic DNA sequence of SEQ ID NO:2
  • b indicates the phage having the genomic DNA sequence of SEQ ID NO:4
  • c indicates the phage having the genomic DNA sequence of SEQ ID NO:5
  • d indicates the phage having the genomic DNA sequence of SEQ ID NO:6
  • e indicates the phage having the genomic DNA sequence of SEQ ID NO:7
  • f indicates the first phage having the genomic DNA sequence of SEQ ID NO:1
  • g indicates the third phage having the genomic DNA sequence of SEQ ID NO:3
  • h indicates the phage having the genomic DNA sequence of SEQ ID NO:8.
  • a indicates the third phage having the genomic DNA sequence of SEQ ID NO: 3
  • b indicates the phage having the genomic DNA sequence of SEQ ID NO: 4
  • c indicates the phage having the genomic DNA sequence of SEQ ID NO: 5
  • d indicates the phage having the genomic DNA sequence of SEQ ID NO: 6
  • e indicates the phage having the genomic DNA sequence of SEQ ID NO: 7
  • f indicates the first phage having the genomic DNA sequence of SEQ ID NO: 1
  • g indicates the second phage having the genomic DNA sequence of SEQ ID NO: 2
  • h indicates the phage having the genomic DNA sequence of SEQ ID NO: 8.
  • "/" indicates that the phages before and after it are used in combination.
  • a/f indicates that the third phage and the first phage are used in combination.
  • 1 is a graph showing the results of a test of disease control effect against bacterial spot of tomato, tested in Example 7. The average disease incidence is shown as a relative value to the untreated group. Graph showing the results of the disease control effect test for broccoli black rot tested in Example 8. The average disease incidence is shown as a relative value to the untreated group. The values shown in each bar graph indicate the type of phage used.
  • a indicates the third phage having the genomic DNA sequence of SEQ ID NO: 3
  • b indicates the phage having the genomic DNA sequence of SEQ ID NO: 9
  • c indicates the phage having the genomic DNA sequence of SEQ ID NO: 8
  • d indicates the phage having the genomic DNA sequence of SEQ ID NO: 10
  • e indicates the phage having the genomic DNA sequence of SEQ ID NO: 5
  • f indicates the phage having the genomic DNA sequence of SEQ ID NO: 6
  • g indicates the phage having the genomic DNA sequence of SEQ ID NO: 7
  • h indicates the phage having the genomic DNA sequence of SEQ ID NO: 11.
  • "/" indicates that the phages before and after it are used in combination.
  • a/b indicates that the third phage and the phage having the genomic DNA sequence of SEQ ID NO: 9 are used in combination.
  • the first aspect of the present invention is a lytic agent.
  • the lytic agent of the present invention comprises a bacteriophage having a genome sequence including a specific base sequence.
  • the bacteriolytic agent of the present invention exhibits specific bacteriolytic activity against target bacteria that may be pathogenic bacteria causing plant diseases.
  • lytic agent refers to an agent consisting of a bacteriophage that has lytic activity against target bacteria.
  • Bacteria are one of the major lineages of organisms that, along with Archaea and Eukaryotes, divide the entire kingdom of life into three parts. Bacteria are made up of cells without a nucleus, and can replicate themselves if they have a source of nutrition. Based on the International Code of Bacteriological Nomenclature, bacteria are named by family, genus, and species.
  • target bacterium refers to a host bacterium that can be targeted by the phage constituting the bacteriolytic agent of the present invention, or the phage contained in the composition and plant disease control composition of the present invention.
  • it is a bacterium that has a membrane surface receptor on the outer cell membrane that is recognized by the phage.
  • the "membrane surface receptor” is the site where, for example, the tail and tail fibers of the phage bind, and is composed of proteins, lipopolysaccharides, pili, etc. present in the outer layer of the bacterial outer membrane.
  • a specific example of the target bacterium in this specification is a bacterium of the genus Xanthomonas.
  • Xanthomonas bacteria are bacteria belonging to the genus Xanthomonas. Xanthomonas bacteria generally produce a yellow pigment called xanthomonadin, and many of them are known to be plant pathogenic bacteria. There are also subspecies and pathovar classifications below species, which are indicated by adding subsp. or pv. respectively after the bacterial name. The smallest unit of classification is the strain, which refers to a group of cells that are considered to be genetically uniform. Table 1 below shows representative Xanthomonas bacteria, their host plants, and the plant diseases they cause.
  • Xanthomonas bacteria Among Xanthomonas bacteria, Xanthomonas arboricola, Xanthomonas citri and Xanthomonas campestris are particularly preferred as target bacteria of the present invention, without being limited thereto.
  • Specific examples of Xanthomonas arboricola include Xanthomonas arboricola pv. pruni, which has the pathotype pruni, and Xanthomonas arboricola pv. juglandis, which has the pathotype juglandis.
  • Specific examples of Xanthomonas citri include Xanthomonas citri subsp. citri.
  • Xanthomonas campestris examples include Xanthomonas campestris pv. vesicatoria, which has the pathotype vesicatoria, Xanthomonas campestris pv. vitians, which has the pathotype vitians, and Xanthomonas campestris pv. campestris, which has the pathotype campestris.
  • Bacteriophage (as mentioned above, in this specification, it is often abbreviated simply to “phage”) is a general term for viruses that infect bacteria.
  • a typical phage is composed of three parts: a head, a tail, and tail fibers.
  • the head is composed of a capsomere, which is an outer coat protein, and is made of a capsid (virus shell) with an icosahedral structure, and contains the phage's genomic DNA in its internal space.
  • the tail has a tubular structure composed of a tail tube protein and a sheath protein that covers it. One end of the tail is connected to the head, and the other end is connected to the tail fiber.
  • the tail functions as an introduction tube that injects the genomic DNA of the head into the cell of the host bacterium.
  • the tail and tail fibers are composed of several fibrous structures made of tail fiber proteins.
  • the tail and tail fibers are responsible for host recognition and adsorption functions, recognizing receptors present on the outer membrane surface of the host bacterium and adsorbing to the cell surface. Phages have extremely high host specificity, a characteristic based on the function of their tails and tail fibers. Because phages do not infect eukaryotes, drugs using phages are harmless to humans, animals, and plants. Phages are broadly classified into “lytic cycle,” “lysogenic cycle,” and “lytic/lysogenic cycle” based on the mode of infection.
  • phages incorporate their own DNA into the bacterial chromosome without lysing the target bacterium, and grow along with the growth of the bacteria.
  • phages grow by themselves within the cells of the host bacterium, then lyse the host bacterium and release large amounts of progeny phages.
  • the phages of the present invention are virulent phages that go through the lytic cycle.
  • tail fiber protein refers to a protein that constitutes the tail fiber of a phage.
  • Tail fiber protein is known to play an important role in the specificity of the host recognition and adsorption ability of the tail and tail fiber (Nobrega F.L. et al., Nat. Rev. Microbiol., 2018, 16:760-773).
  • Tail fiber gene refers to a gene contained in the phage genomic DNA that encodes the tail fiber protein.
  • Tail tube protein is a protein that constitutes the tubular structure of the tail of a phage. It is known that the tail tube protein interacts with the tail fiber and, together with the tail fiber, plays an important role in the specificity of host recognition and adsorption ability (Maozhi Hu, et al., 2020, 9:1, 855-867).
  • Tail tube proteins include tail tube fiber protein A and tail tube protein B.
  • Tail tube protein A is a protein that forms a ring at the lower part of the tubular structure of the tail and interacts with the tail fiber.
  • “Tail tube protein B” is a protein that forms the lower end of the tubular structure of the tail and binds to a receptor present on the outer membrane surface of the host bacterium.
  • Tiil tube gene refers to a gene contained in the phage genomic DNA that codes for the tail tube protein.
  • Tiil tube protein A gene refers to the gene that codes for tail tube protein A
  • tail tube protein B gene refers to the gene that codes for tail tube protein B.
  • Bacteriolysis refers to the phenomenon of destroying the bacterial cell membrane. As mentioned above, this phenomenon is mainly seen in the infection mode of virulent phages. Bacteria die as a result of bacteriolysis. Bacteriolysis begins when a phage specifically adsorbs to a target bacterium and injects its own DNA into the target bacterium's cell via its tail. The phage then uses the bacterial translation mechanism to replicate itself and produce a large amount of progeny phages, which are then lysed and released into the outside world.
  • plant disease refers to a general term for illnesses that occur in plants.
  • Known plant diseases include diseases caused by infectious pathogens such as viruses, bacteria, fungi, actinomycetes, viroids, phytoplasmas, nematodes, mites, or insects, as well as diseases caused by non-infectious pathogens such as a lack or excess of nutrients or water, or chemical damage.
  • plant diseases in this specification refer to diseases caused by bacteria, i.e., plant pathogenic bacteria.
  • plant pathogenic bacteria in this specification refer to the aforementioned target bacteria, for example, Xanthomonas bacteria.
  • control refers to prevention or treatment (eradication) (from the Japan Pesticide Manufacturers Association website). Therefore, in this specification, “plant disease control” refers to the prevention of plant diseases, particularly target bacteria, or the treatment of plant diseases caused by target bacteria.
  • target plant refers to a plant to which the plant disease control composition of the present invention described below is applied. This plant corresponds to a plant that has developed a specific plant disease due to infection with the target bacterium, or a plant that is at risk of infection with the target bacterium.
  • the lytic agent of the present invention comprises a bacteriophage.
  • the first phage is characterized by having a genomic DNA sequence that includes a specific base sequence, and the lytic agent of the present invention exhibits specific lytic activity against a target bacterium.
  • the first phage has a genome DNA sequence that is identical to the 42,933 bp base sequence shown in SEQ ID NO: 1, or the base sequence shown in SEQ ID NO: 1 in which one or more bases have been added, deleted, and/or substituted, or has a similar affinity to the base sequence shown in SEQ ID NO: 1 by 92.15% or more, 92.2% or more, 92.3% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 99% or more, 10 ...
  • the base sequence has a sequence identity of 8.5% or more, 99% or more, or 99.5% or more, a base sequence having a sequence identity of 99.01% or more, 99.05% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more when aligned with the base sequence shown in SEQ ID NO: 1, and further has a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 1.
  • the range to be compared is 100% of the entire genome of the first phage, but it may also be 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93.5% or more, or 93% or more.
  • multiple refers to 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.
  • (base) sequence identity is a numerical value indicating the proportion of sites with the same type of base within the comparison range of two base sequences. Even if the lengths of the two base sequences are different, the base sequence identity can be calculated by aligning the sequences so that the base match within the comparison range is the highest.
  • BLAST can be used in various software and web services. For example, the genetic information processing software GENETYX (https://www.genetyx.co.jp/) and the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi) can be used to easily calculate the base sequence identity.
  • GENETYX https://www.genetyx.co.jp/
  • NCBI BLAST server https://blast.ncbi.nlm.nih.gov/Blast.cgi
  • FASTA an algorithm that can be used as long as it can calculate a reasonable identity, and there is no particular limitation on the method used.
  • highly stringent conditions refer to environmental conditions that make it difficult for non-specific hybridization to occur. Under highly stringent conditions, a hybrid can be formed with a nucleic acid having a target base sequence, but a hybrid cannot be substantially formed with a nucleic acid having a non-specific base sequence.
  • highly stringent conditions refer to conditions of low salt concentration and high temperature.
  • a low salt concentration is, for example, 15 mM to 750 mM, preferably 15 mM to 500 mM, 15 mM to 300 mM, or 15 mM to 200 mM.
  • a high temperature is, for example, 50 to 68°C, or 55 to 70°C.
  • a specific example of highly stringent conditions is washing after hybridization at 65°C with 0.1xSSC and 0.1% SDS.
  • a phage is a virus, and it is conceivable that mutations such as substitutions, deletions, and insertions may occur in the genomic DNA when amplifying the phage of the present invention. As long as the degree of such mutations is within the ranges described above for the entire genomic DNA and the lytic function of the phage is maintained, the present invention includes such mutations.
  • the average nucleotide identity may be used as an index of sequence identity, and these may also be used. It is not easy to align and compare the entire range of long phage genome DNA. Therefore, the above sequence identity may be obtained within the range automatically aligned by the above software or web service. For example, in an analysis using the BLAST server provided by NCBI, the query sequence and subject sequence are automatically aligned (often referred to as "aligned” in this specification) to the maximum possible range, and the comparison range is determined. The sequence identity within this comparison range is calculated, and the ratio of the comparison range to the entire range of the query sequence may be calculated as a value called Query Cover.
  • sequence identity of the base sequences in the entire range of the aligned base sequences can be estimated based on the results.
  • the Query Cover value multiplied by the sequence identity value in the aligned range can be used as an estimate of sequence identity in the entire range.
  • further corrections can be made to improve the accuracy of the estimate, for example by including sequence identity expected in ranges outside the aligned range.
  • phage genomic DNA When phage genomic DNA is packaged, it can be either linear or circular.
  • genomic DNA In next-generation genome sequencer analysis, the genomic DNA is fragmented, the base sequences of the individual fragments are read, and the fragments are then linked together to determine the sequence.
  • the fragments In the case of phages, the fragments are often linked together without a reference genomic DNA sequence (de novo assembly). This makes it difficult to unambiguously determine the start and end of the analyzed genome (Merrill, B.D., et al. BMC Genomics, 2016 17, 679).
  • the start and end of the genome sequences to be compared can be different, and this is automatically taken into account in analysis using software or analysis servers.
  • Xanthomonas bacteria Xanthomonas arboricola, Xanthomonas citri, and Xanthomonas campestris are particularly preferred as target bacteria for the first phage.
  • Specific examples of Xanthomonas arboricola suitable as target bacteria for the first phage include Xanthomonas arboricola pv. pruni, which has the pathotype pruni, and Xanthomonas arboricola pv. juglandis, which has the pathotype juglandis.
  • Specific examples of Xanthomonas citri suitable as target bacteria for the first phage include Xanthomonas citri subsp. citri.
  • Xanthomonas campestris suitable as a target bacterium for the first phage include Xanthomonas campestris pv. vesicatoria, which has the pathotype vesicatoria, and Xanthomonas campestris pv. vitians, which has the pathotype vitians.
  • the lytic agent of the present invention containing the first phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.
  • the second phage is characterized by having a genomic DNA sequence of a specific length containing a specific base sequence, and the lytic agent of the present invention exhibits specific lytic activity against the target bacterium.
  • the second phage has a genomic DNA sequence which is identical to the 35,321 bp nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence shown in SEQ ID NO: 2 in which one or more nucleotides have been added, deleted, and/or substituted, or which has a similar identity to the nucleotide sequence shown in SEQ ID NO: 2 by 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more a base sequence having sequence identity of 99% or more, or 99.5% or more; a base sequence having sequence identity of 95.0% or more, 95.5% or more, 96.0% or more
  • the comparison range (hereinafter sometimes referred to as Query Cover) cover 100% of the entire genome of the second phage, but it may also cover 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, or 93% or more.
  • the total length of the genomic DNA sequence of the second phage is 40,000 bases or less.
  • the total length of the genomic DNA sequence is 40,000 bases or less, 39,500 bases or less, 39,000 bases or less, 38,500 bases or less, 38,000 bases or less, 37,500 bases or less, 37,000 bases or less, 36,500 bases or less, 36,000 bases or less, 35,900 bases or less, 35,800 bases or less, 35,700 bases or less, 35,600 bases or less, 35,500 bases or less, or 35,400 bases or less.
  • Xanthomonas arboricola and Xanthomonas campestris are particularly preferred as target bacteria for the second phage, although there is no particular limitation thereto.
  • Specific examples of Xanthomonas arboricola suitable as target bacteria for the second phage include Xanthomonas arboricola pv. pruni, which has the pathotype pruni, and Xanthomonas arboricola pv. juglandis, which has the pathotype juglandis.
  • Specific examples of Xanthomonas campestris suitable as target bacteria for the second phage include Xanthomonas campestris pv.
  • vesicatoria which has the pathotype vesicatoria
  • Xanthomonas campestris pv. vitians which has the pathotype vitians
  • Xanthomonas campestris pv. campestris which has the pathotype campestris.
  • the lytic agent of the present invention containing the second phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.
  • the third phage is characterized by having a genomic DNA sequence containing a specific base sequence, and the lytic agent of the present invention exhibits specific lytic activity against the target bacterium.
  • the third phage has a genomic DNA sequence which is identical to the 43,601 bp nucleotide sequence shown in SEQ ID NO: 3, or the nucleotide sequence shown in SEQ ID NO: 3 in which one or more nucleotides have been added, deleted, and/or substituted, or which is different from the nucleotide sequence shown in SEQ ID NO: 3 by 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more,
  • the base sequence has a sequence identity of 98% or more, 98.5% or more, 99% or more, or 99.5% or more; a base sequence having a sequence identity of 96.9% or more, 97.0% or more, 97.
  • the comparison range (hereinafter sometimes referred to as Query Cover) is 100% of the entire genome of the third phage, but it may also be 99% or more, 98% or more, 97% or more, 96% or more, 95% or more, 94% or more, 93% or more, 92% or more, 91% or more, 90% or more, 89% or more, or 88% or more.
  • Xanthomonas arboricola and Xanthomonas campestris are preferred as target bacteria for the third phage, without being particularly limited thereto.
  • Specific examples of Xanthomonas arboricola suitable as target bacteria for the third phage include Xanthomonas arboricola pv. juglandis, which has a pathotype of juglandis.
  • Specific examples of Xanthomonas campestris suitable as target bacteria for the third phage include Xanthomonas campestris pv.
  • vesicatoria which has a pathotype of vesicatoria
  • Xanthomonas campestris pv. vitians which has a pathotype of vitians
  • Xanthomonas campestris pv. campestris which has a pathotype of campestris.
  • the lytic agent of the present invention containing the third phage can exhibit lytic activity against bacteria of the genus Xanthomonas and can be applied to plant diseases.
  • the second aspect of the present invention is a composition, particularly a composition that can be used for controlling plant diseases.
  • the composition of the present invention is characterized by containing the lytic agent according to the first aspect as an active ingredient.
  • composition of the present invention when used to control plant diseases, can provide a sustainable pesticide against bacterial plant diseases that is safe for the human body, has no chemical hazard to the environment, and can specifically prevent or treat the target plant disease.
  • plant disease control composition refers to the use of the composition of the present invention for plant disease control purposes.
  • composition 2-2-1 Constituents
  • the composition of the present invention contains, as an essential constituent, the bacteriophage that is the lytic agent according to the first aspect as an active ingredient. It may also contain an agriculturally acceptable carrier and/or medium within a range that does not inhibit or suppress the lytic activity of the phage against the target bacterium. If necessary, it may further contain other active ingredients. Each of the constituents will be specifically described below.
  • the composition of the present invention contains the lytic agent according to the first aspect as an essential active ingredient.
  • the target bacterium of the present invention is lysed by this active ingredient, and therefore plant diseases caused by the target bacterium can be prevented or treated.
  • the amount of active ingredient contained per unit amount in the composition depends on various conditions such as the formulation, type of plant pathogenic bacteria, type of target plant, application location, and application method. It is preferable that the phage, which is the active ingredient, is contained in an amount sufficient to contact and infect the plant pathogenic bacteria that have infected the target plant. Therefore, it is sufficient to take into consideration each condition and determine, within the scope of common technical knowledge in the field, that the lytic agent contained in the plant disease control composition of the present invention is in an effective amount against the target bacteria after application.
  • Agriculturally acceptable carriers and media refers to substances that facilitate application of the composition, can maintain the viability and infectivity of the active ingredient, the phage, and/or can control the rate of action, and that have no or very little harmful effects on the environment, such as soil and water quality, when applied outdoors, and further have no or very little harmful effects on animals, especially humans.
  • Carrier Specific examples of agriculturally acceptable carriers include surfactants, protective agents, excipients, etc. If desired, small amounts of wetting agents, emulsifiers, pH buffers, etc. may also be used. The carrier may be mixed in advance or immediately before application.
  • Surfactants have the effect of improving the physicochemical properties of the composition, such as wetting, emulsifying, dispersing, penetrating, adhering, defoaming, and spreading properties of the composition on plant bodies.
  • Surfactants can be used as the main component of agricultural chemical adjuvants called spreading agents.
  • spreading agents include nonionic surfactants, combinations of nonionic and anionic surfactants, paraffin-based surfactants, and polyoxyethylene resin acid esters.
  • More specific examples include polyoxyethylene alkyl ether compounds, polyoxyethylene fatty acid ester compounds, lignin sulfonate compounds, naphthylmethanesulfonate compounds, alkylsulfosuccinate compounds, and tetraalkylammonium salt compounds.
  • protective agents are expected to have the effect of reducing damage caused by ultraviolet rays.
  • examples include skim milk, casein, gelatin, etc.
  • excipients examples include glucose, lactose, sucrose, gelatin, starch, malt, and wheat flour.
  • solvents include water (including aqueous solutions), buffers, and liquid media.
  • the solvent is preferably a sterile liquid.
  • composition of the present invention may contain one or more other active ingredients having the same and/or different pharmacological action as long as they do not affect the lytic activity of the phage constituting the lytic agent.
  • the type of other active ingredient is not important.
  • it may be a phage that has lytic activity against the same and/or different bacteria.
  • phages that have lytic activity against the same bacteria include other phages that specifically recognize and bind to Xanthomonas bacteria, similar to the phages that constitute the lytic agent described in the first embodiment.
  • a synergistic or complementary effect of lytic activity can be expected by combining them.
  • phages that specifically recognize such Xanthomonas bacteria and have lytic activity are not particularly limited. Specific examples include phages whose genomic DNA contains the base sequence shown in any one of SEQ ID NOs: 4 to 11, and mutants and modified forms thereof.
  • the plant disease control composition of the present invention can contain, in addition to the lytic agent described in the first embodiment, one or more other phages that specifically recognize the Xanthomonas bacteria specifically exemplified above and have lytic activity in combination as active ingredients.
  • sequence identity of the amino acid sequences is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • “multiple” refers to 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.
  • amino acid substitution refers to substitution within a conservative amino acid group that has similar properties such as charge, side chain, polarity, and aromaticity among the 20 types of amino acids that make up natural proteins. Examples include substitutions within the uncharged polar amino acid group with low polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of polypeptides.
  • amino acid sequence identity is a numerical value indicating the percentage of sites in which the types of amino acid residues are the same within the comparison range of two amino acid sequences. Even if the lengths of the two amino acid sequences are different, amino acid sequence identity can be calculated by aligning the sequences so that the degree of amino acid identity within the comparison range is maximized.
  • the algorithm used to perform such an analysis can be one already mentioned, such as BLAST.
  • active ingredients may include insecticides, herbicides, fertilizers (e.g., urea, ammonium nitrate, superphosphates), and, if desired, known chemical pesticides, antibiotics, and biological pesticides.
  • fertilizers e.g., urea, ammonium nitrate, superphosphates
  • chemical pesticides antibiotics, and biological pesticides.
  • composition of the present invention may be in any form, as long as it is capable of maintaining the infection site of the target bacterium on the target plant, the fixation to the target plant, and/or the ease of infection of the phage, which is the active ingredient, to the target bacterium, when used as a plant disease control composition.
  • the plant disease control composition may be a liquid or wettable powder in a liquid state suspended in a suitable solution, or may be mixed with a carrier and solidified into a solid powder, granule, or gel.
  • the infection site of the target bacterium in the target plant is the leaves, flowers, fruits, stems, branches, or trunks of the aboveground part
  • a liquid, wettable powder, or gel formulation that is widely distributed to the infection site and has high fixation is suitable, although not limited thereto.
  • the infection site of the target bacterium is the roots or rhizomes of the underground part
  • a powder or granule that is slowly released in the soil and can exert a sustained effect on the infection site is suitable, although not limited thereto.
  • the application method may be any method known in the art, as long as it can apply the plant disease control composition to the target plant, and is not particularly limited.
  • the phage which is the active ingredient of the plant disease control composition, can invade the entire surface of the plant body, such as the stems and leaves and roots of the target plant, and therefore can be applied in a suitable manner according to the purpose.
  • the application site is an above-ground part such as the stems and leaves
  • the plant disease control composition may be applied so as to directly contact the application site. Examples of direct contact include coating, spraying, scattering, or immersion of the plant disease control composition on the application site.
  • the composition may be applied to a site of the target plant that is infected by the target bacterium or a site that may be infected. If the application site is an underground part such as the roots, the composition may be added to the soil, or if the application site is a medium, the composition may be added to the medium indirectly.
  • the "soil” referred to here is not particularly limited as long as it is soil in which the target plant can grow. Usually, a soil for planting containing appropriate nutrients and having an appropriate pH value is used. The location of the soil does not matter.
  • the "culture medium” refers to an artificially prepared culture medium for planting the target plant. It may be a solid culture medium such as an agar medium, or a liquid culture medium. Examples of the culture medium include an isolation bed, a root-zone restricted pot, or a seedbed.
  • the composition of the culture medium may be a culture medium composition known in the art. It can be appropriately selected depending on the type of plant, etc.
  • Target plants The target plant of the plant disease control composition of the present invention is not particularly limited in type, so long as it is a plant that can develop a plant disease caused by the target bacterium of the present invention. It may be either an angiosperm or a gymnosperm. Furthermore, it does not matter whether it is a herbaceous plant or a woody plant. Suitable specific examples of target plants include agriculturally important plants, such as crop plants such as cereals, vegetables, and fruits, and ornamental plants.
  • monocotyledonous plants include plants of the Poaceae family (e.g., rice, wheat, barley, corn, sugarcane, sorghum, sorghum, and turfgrass), plants of the Musaceae family (e.g., banana), plants of the Amaryllidaceae family (e.g., leeks, onions, garlic, and Chinese chives), and plants of the Liliaceae family (e.g., lilies and tulips).
  • the Poaceae family e.g., rice, wheat, barley, corn, sugarcane, sorghum, sorghum, and turfgrass
  • plants of the Musaceae family e.g., banana
  • plants of the Amaryllidaceae family e.g., leeks, onions, garlic, and Chinese chives
  • plants of the Liliaceae family e.g., lilies and tulips.
  • dicotyledonous plants include Brassicaceae plants (e.g., cabbage, radish, Chinese cabbage, rapeseed), Asteraceae plants (e.g., lettuce, burdock, chrysanthemum), Fabaceae plants (e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, licorice), Solanaceae plants (e.g., tomato, eggplant, potato, tobacco, bell pepper, capsicum, petunia), and Rosaceae plants (e.g., strawberry, apple, pear, peach, loquat, almond, plum, rose, plum, Japanese plum, etc.).
  • Brassicaceae plants e.g., cabbage, radish, Chinese cabbage, rapeseed
  • Asteraceae plants e.g., lettuce, burdock, chrysanthemum
  • Fabaceae plants e.g., soybean, peanut, pea, kidney bean, lentil, chickpe
  • Cucurbitaceae plants e.g., cucumber, gourd, pumpkin, melon, watermelon
  • Anacardiaceae plants e.g., mango, pistachio, cashew nut
  • Lauraceae plants e.g., avocado
  • Rutaceae plants e.g., mandarin orange, grapefruit, lemon, yuzu
  • Convolvulaceae plants e.g., sweet potato
  • Theaceae plants e.g., tea plant
  • Vitaceae plants e.g., grapes.
  • Target plant diseases to which the plant disease control composition of the present invention is applied include all plant diseases caused by the target bacteria of the present invention.
  • the plant diseases are caused by Xanthomonas bacteria.
  • the plant diseases include bacterial spot disease found in peaches, bacterial blight disease found in walnuts, bacterial pustule disease found in soybeans, angular leaf spot disease found in strawberries, bacterial blight disease found in tomatoes, peppers, lettuce, black rot disease found in cabbage, Chinese cabbage, broccoli, bacterial canker disease found in oranges, grapefruits, angular leaf spot disease found in cotton, and bacterial leaf blight disease found in rice.
  • Plant disease control method 3-1 Overview
  • the third aspect of the present invention is a plant disease control method.
  • the plant disease control method of the present invention is characterized in that the plant disease control composition according to the second aspect is applied to a target plant to control the plant disease of the target plant.
  • the plant disease control method of the present invention makes it possible to control plant diseases caused by bacteria, particularly Xanthomonas bacteria, in target plants.
  • the plant disease control method of the present invention includes a contact step as an essential step.
  • the "contact step” refers to a step of contacting a target plant with the plant disease control composition described in the second embodiment. This step basically conforms to "2-3. Application method" for the plant disease control composition of the second embodiment.
  • contact refers to contact between the plant disease control composition and the target plant. More specifically, it refers to contact of the lytic agent described in the first embodiment, which is the active ingredient of the plant disease control composition, i.e., the phage, with the plant body of the target plant, preferably a site infected or at risk of infection with the target bacterium.
  • the purpose of this step is to infect the target bacterium with the phage, which is the active ingredient, thereby lysing the target bacterium. As a result, a control effect against plant diseases caused by the target bacterium can be exerted.
  • the contact may be either direct or indirect.
  • direct contact refers to the plant disease control composition coming into direct contact with a specific part of the target plant.
  • the plant body of the target plant is coated, sprayed, sprinkled or immersed in a liquid or gel plant disease control composition.
  • the plant body that is the subject of contact is mainly the leaves, flowers, fruits, stems, branches and/or trunks.
  • indirect contact in this embodiment refers to the plant disease control composition coming into contact with a specific part of the target plant via an intermediary.
  • indirect contact refers to the application of a granular plant disease control composition in the soil around the roots of the target plant.
  • the phage which is the active ingredient, is transported via water in the soil and is eventually absorbed by the roots.
  • the phage which is an active ingredient of the composition obtained by the present invention, can efficiently kill the target bacteria, and is therefore useful for preventing and suppressing diseases caused by the target bacteria.
  • the detection and identification of the target bacteria based on its specific bacteriolytic activity makes it possible to diagnose the disease.
  • Copper agents and antibiotics are examples of drugs that have been conventionally used against Xanthomonas bacteria, but these drugs can cause phytotoxicity and disrupt the balance of the microflora.
  • the fourth aspect of the present invention is a method for identifying bacteria belonging to the genus Xanthomonas.
  • the identification method of the present invention is characterized in that it identifies bacteria belonging to the genus Xanthomonas by utilizing the host specificity of the phage constituting the lytic agent described in the first aspect.
  • the present invention makes it possible to determine whether or not an unidentified plant pathogenic bacterium causing a plant disease is a bacterium of the genus Xanthomonas, and to identify it.
  • the identification method of the present invention includes a culturing step, a mixing step, a mixture culturing step, and a determination step as essential steps, and an isolation step as a selection step. Each step will be described below.
  • Isolation step is a step of isolating a test bacterium from a plant tissue infected with a plant disease. This step is a selection step and may be performed as necessary.
  • test bacterium refers to a plant pathogenic bacterium that is subjected to the Xanthomonas bacteria identification method of the fourth aspect of the present invention, and whose species has not been identified.
  • the plant tissue may be any part of the plant that has a plant disease, but preferably a part where the symptoms of the plant disease are clearly visible. For example, if the plant is a peach that has bacterial peach hole, the leaf where the disease is visible may be used.
  • the diseased specimen can be immersed in a solvent such as water and extracted, and the specimen can be fragmented or crushed during extraction. The extract can then be streaked onto an agar medium and a single colony picked.
  • the "culturing step” is a step of culturing the isolated test bacterium to obtain a culture.
  • the method for culturing the test bacterium may be a method known in the art.
  • a "culture” is something obtained by culturing a test bacterium, and may be either liquid or solid.
  • test bacteria are unidentified, so it is desirable to use a medium capable of culturing a wide variety of plant pathogenic bacteria.
  • a medium capable of culturing at least Xanthomonas bacteria which are the bacteria to be identified in the present invention, is used.
  • Such a medium may contain one or more components selected from, for example, protein enzyme hydrolysates such as peptone and tryptone, biological extracts such as potato dextrose and yeast extract, amino acids such as glutamic acid or salts thereof, sugars such as glucose and sucrose, and inorganic salts such as sodium chloride, magnesium chloride, and potassium dihydrogen phosphate.
  • Specific media and compositions include LB medium (tryptone, yeast extract, sodium chloride), YPG medium (yeast extract, peptone, glucose), PD medium (potato dextrose), and Suwa medium with added peptone (sucrose, glutamic acid, peptone).
  • the isolated test bacteria are seeded on the medium and cultured under appropriate culture conditions.
  • a culture can be obtained by culturing at 20-40°C, 20-30°C, 22-28°C, or 24-26°C with stirring. There are no limitations on the culture time, but it is sufficient to culture until the turbidity at a wavelength of 600 nm reaches about 1.0. This process produces a culture solution of the test bacteria.
  • the culture may also be a multi-stage culture of two or more stages. For example, a soft agar-containing liquid medium can be added to the culture solution obtained after culture in a liquid medium, poured onto a solid medium such as an agar medium to solidify, and then further cultured.
  • the “mixing step” is a step of mixing the culture obtained in the culturing step with the lytic agent according to the first aspect to obtain a mixture.
  • the “mixture” refers to a mixture of a culture and a lytic agent, and may be either liquid or solid.
  • the lytic agent described in the first embodiment may be in a solid state, or may be administered in a liquid state suspended in water or a liquid medium.
  • the volume ratio of culture to lytic agent may be 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1.
  • the culture and lytic agent may be mixed thoroughly by stirring or the like.
  • the lytic agent may be dropped onto a solid culture such as a gel surface, and the two may be mixed on the solid medium to obtain a mixture.
  • the "mixture cultivation step” is a step of culturing the mixture under predetermined conditions.
  • a soft agar-containing liquid medium can be added to the mixture, poured onto a solid medium such as an agar medium, allowed to solidify, and then further cultured.
  • the basic procedure of this step is similar to that of the culturing step described above.
  • this step although not limited thereto, it is preferable to carry out culturing based on the so-called plaque assay method so as to easily confirm whether or not the phage constituting the lytic agent has lysed the test bacterium in the determination step described below.
  • a part of the mixture is mixed with a soft agar medium of the same composition, and then, before the soft agar medium solidifies, it is poured onto an agar medium of the same composition and spread over the entire medium. Then, the mixture is cultured under the same conditions as in the culturing step described above.
  • the "determination step” is a step of determining that the test bacterium is a bacterium of the genus Xanthomonas when the test bacterium is lysed after the culture step.
  • the determination of the presence or absence of bacteriolysis may be made based on the presence or absence of plaque formation. If plaque is present on the solidified soft agar medium spread on the agar medium after the above-mentioned mixture culture process, this indicates that the test bacterium has been lysed by infection with the phage that constitutes the bacteriolytic agent of the present invention. Therefore, the test bacterium in this case can be determined to be a bacterium of the genus Xanthomonas. On the other hand, if the test bacterium grows over the entire agar medium and no plaque is present at all, the test bacterium can be determined to not be a bacterium of the genus Xanthomonas.
  • a negative control may be prepared by mixing with a medium that does not contain a lytic agent during the mixture culture process, and/or a positive control may be prepared by using identified Xanthomonas bacteria from the culture process instead of the test bacteria, and it may be confirmed that no plaques are formed in the negative control and that plaques are observed in the positive control.
  • the method for identifying Xanthomonas bacteria of the present invention makes it possible to detect whether or not Xanthomonas bacteria are present in the lesions of a plant that has developed a plant disease that is suspected to be caused by Xanthomonas bacteria.
  • Example 1 Isolation of a novel bacteriophage and its lytic activity (1) (the purpose) We will isolate novel bacteriophages that have lytic activity against pathogenic bacteria that cause plant diseases, and verify their lytic activity against plant pathogenic bacteria.
  • NCIMB Newcastle disease virus
  • a liquid medium (YPG Broth) was used, which was prepared by dissolving 1 g of peptone, 1 g of yeast extract, and 2 g of glucose in 1 L of H2O and autoclaving.
  • an agar medium was used in which 15 g of agar per 1 L of the above-mentioned broth (YPG Broth) was added and autoclaved (when YPG Broth was used, it was written as "YPG Agar").
  • Each of the above strains delivered in a dry powder form, was suspended in 0.1 mL of broth and then streak cultured on agar (YPG agar) at 25°C to isolate single colonies.
  • the isolated colonies were inoculated into broth and cultured with shaking at 25°C to serve as preculture medium.
  • the preculture medium was inoculated into broth and cultured at 25°C for 10 to 30 hours until the turbidity (Optical Density 600 nm) reached approximately 1.0.
  • the culture medium after cultivation was used as is as the bacterial liquid.
  • the novel phage was isolated from natural wastewater or soil obtained in Japan.
  • the phage was isolated based on the conventional plaque assay method.
  • wastewater from ponds or lakes, or wastewater obtained by suspending soil in water was filtered through a 0.45 ⁇ m filter to prepare a phage-containing liquid.
  • Equal amounts of the bacteria liquid and the phage-containing liquid were then mixed and left at room temperature for about 10 minutes.
  • 0.2 mL of the bacteria/phage mixture was added to 3 mL of Top Agar, quickly mixed in a vortex mixer, and then poured onto the agar. After the Top Agar solidified, it was left to stand at 25°C for about 12 hours.
  • a lytic plaque was formed on the lawn of bacteria formed by the culture. Then, the gel of the plaque portion was aspirated using a cut-off tip, and a phage having lytic activity against Xanthomonas bacteria was isolated. The phage was then purified by repeating this procedure using a phage-containing liquid containing a high concentration of the isolated phages instead of the wastewater.
  • the new phage isolated in Example 1 is referred to as the "first phage.”
  • the isolated phages were suspended in SM Buffer and passed through a 0.2 ⁇ m filter to recover the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to further purify the phages.
  • the composition of the SM Buffer is shown in Table 3.
  • the plate lysate (PL) method which is an amplification method using a plaque assay method, was carried out.
  • a bacteria/phage mixture was prepared so that many plaques would form on the agar, and then mixed with Top Agar, and spread on YPG Agar and cultured.
  • 3 mL of SM Buffer was added to the Top Agar on which the plaques had formed, and the mixture was shaken at 25°C for about 30 minutes, and the supernatant was passed through a 0.2 ⁇ m filter to recover the recovery liquid containing the phage.
  • the first phage purification was carried out by adding 1 g of PEG 6000 (final concentration 10%) and 0.4 g of NaCl (final concentration 4%) to 10 mL of the collected solution, dissolving the solution, and rotating the solution overnight at 4°C using a rotator. The solution was then centrifuged at 15,000g/4°C/60 minutes, and the supernatant was removed. The collected pellet was resuspended in 0.5 mL of SM Buffer. Then, 0.5 mL of chloroform was added, vigorously stirred, and left on ice for 6 hours. After centrifugation at 8,000g/4°C/10 minutes, the upper layer was carefully collected to obtain a phage purified solution.
  • the concentration of the phage purified solution is generally expressed as a titer [PFU/mL] based on the number of plaques (Plaque Forming Unit, PFU) in a plaque assay, which is an index of bacteriolytic activity.
  • PFU Protein Forming Unit
  • the titer of the prepared first phage purified solution was determined by a plaque assay method using an appropriately diluted solution, and was confirmed to be 10 8 PFU/mL or more.
  • the host range of the first phage was evaluated by the spot test method. 0.1 mL of the bacterial solution was added to 3 mL of Top Agar, mixed, and poured onto the agar, and the solution was spread over the entire plate and solidified. In addition to the bacterial solutions of each Xanthomonas bacterium shown in Table 2 prepared in (1) above, a bacterial solution of Pseudomonas fluorescens for control was also prepared. Then, about 5 ⁇ L of the purified phage solution was dropped and incubated at 25 ° C for about 12 hours. If the dropped area on the plate on which the lawn of bacteria was formed became clear in a circle (diameter about 1 cm), it was determined that the dropped phage had bacteriolytic activity against that strain.
  • first phage exhibits lytic activity
  • only the area of the lawn of bacteria formed on the plate where the phage purification liquid was dropped becomes transparent.
  • One type of first phage obtained by the present invention exhibited lytic activity against all strains belonging to the three species of bacteria shown in Table 2, namely, Xanthomonas arboricola (including pv. pruni and pv. juglandis), Xanthomonas campestris (including pv. vesicatoria and pv. vitians), and Xanthomonas citri.
  • the first phage may be applicable to various plant diseases. For example, it is suggested that it may be useful in controlling peach bacterial hole, walnut bacterial black spot, tomato bacterial spot, lettuce bacterial spot, and citrus canker, which are caused by Xanthomonas bacteria, and it is expected that it will also be of great value in industrial applications.
  • Genome Analysis of the First Phage The genomic DNA sequence of the first phage was determined and analyzed.
  • the concentration of the genomic DNA was measured using a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific), and 50 ⁇ L of the genomic DNA solution was prepared so that the final concentration was 0.2 ng/ ⁇ L.
  • the genome of the first phage was fragmented and an adapter sequence was added by PCR using Nextera XT DNA Library Prep (Illumina) according to the attached manual.
  • electrophoresis was performed using a Bioanalyzer (Agilent Technologies) with an Agilent High Sensitivity DNA Kit (Agilent Technologies), the average bp size of the sample was measured, and the concentration of the DNA fragments was calculated.
  • a measurement sample was prepared using a Miseq Reagent kit (Illumina) by processing according to the attached manual, and measurements were performed using a next-generation sequencer Miseq (Illumina). After preprocessing (trimming, etc.) of the obtained data using CLC genomics workbench (Qiagen), de novo assembly was performed to obtain a contig sequence corresponding to the phage genome sequence.
  • Miseq Reagent kit Illumina
  • CLC genomics workbench Qiagen
  • sequence with the highest sequence identity was the genome DNA sequence of Pseudomonas phage vB_Pae_TR (access code: OL802211.1), which had a sequence identity of 99.07% over a range of 93% of the full length of SEQ ID NO: 1.
  • the sequence identity is a numerical value for the range that the analysis server automatically aligned against the entire genome length of the first phage.
  • the numerical value for that range is displayed as Query Cover, and the sequence identity of vB_Pae_TR, 99.07%, is a numerical value calculated by limiting it to a region that accounts for 93% of the entire length of ⁇ X-33. Therefore, while it is difficult to calculate the sequence identity for the entire length, it is estimated that the numerical value will be at least lower than 99.07%, and usually corresponds to a numerical value equal to or lower than 93%.
  • the target bacteria of phage vB_Pae_TR which has a genome with the above-mentioned publicly known sequence, was revealed to be Pseudomonas bacteria. Therefore, it can be said that it was difficult to identify the target bacteria of the first phage isolated in this example based on sequence identity with the genome sequence of the publicly known phage.
  • a phage with a higher sequence identity over its entire length than vB_Pae_TR (a phage showing higher sequence identity over a wider range) is a phage with a similar host range to the first phage isolated in this example, that is, a phage that uses a variety of Xanthomonas bacteria as host bacteria.
  • Example 2 Isolation of a novel bacteriophage and its lytic activity (2) (the purpose) We will isolate novel bacteriophages that have lytic activity against pathogenic bacteria that cause plant diseases, and verify their lytic activity against plant pathogenic bacteria.
  • NCIMB-ID 10460
  • Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Obtaining and culturing plant pathogenic bacteria" in Example 1.
  • the isolated phages were suspended in SM Buffer and passed through a 0.2 ⁇ m filter to recover the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.
  • a plate lysate (PL) method was carried out to amplify and purify the isolated and purified second phage.
  • the specific method was in accordance with the method described in "(3) Amplification and purification of phage" in Example 1.
  • the titer of the prepared second phage purified solution was determined by a plaque assay method using an appropriately diluted solution, and was confirmed to be 10 8 PFU/mL or more.
  • the second phage is also likely to be applicable to a variety of plant diseases, and these results suggest that it may be useful for controlling, for example, peach bacterial hole, walnut bacterial black spot, tomato bacterial spot, and lettuce bacterial spot, which are caused by Xanthomonas bacteria.
  • Genome Analysis of the Second Phage The genomic DNA sequence of the second phage was determined and analyzed.
  • Xanthomonas oryzae is known as a bacterial species in which phage Xp12 exhibits bacteriolytic activity (Nakayinga R. et al., BMC Microbiology, 2021, 21:291).
  • the total length of the genomic DNA sequence of Xp12 is 63,783 bases, while the total length of the genomic DNA sequence of the second phage isolated in this example is 35,321 bases, which is about half the total length.
  • sequence identity when the sequence identity when the genomic DNA sequence of Xp12 is used as a standard was calculated using GENETYX-NGS implemented in the genetic information processing software GENETYX (https://www.genetyx.co.jp/), the base sequence of the genomic DNA of the second phage (SEQ ID NO: 2) had a sequence identity of 98.34% over a range of 54% of the total length of the genomic DNA sequence of Xp12.
  • the sequence identity when comparing the total lengths of the genomic DNA sequences of both phages was estimated to be about 53.10%. From this, it can be said that the genomic DNA sequence of the second phage obtained in this example only has a sequence homologous to a part of the genomic DNA sequence of Xp12, and the overall similarity is not high.
  • genomic DNA sequences of Xp12 and the phage of the present invention were compared using MUMmer implemented in the genetic information processing software GENETYX (https://www.genetyx.co.jp/).
  • GENETYX https://www.genetyx.co.jp/.
  • the region contained a gene (access code: QNN97189.1) that was presumed to be a tail fiber gene based on the sequence identity, a tail fiber gene (access code: QNN97196.1), and an endolysin gene (access code: QNN97201.1). These genes are deeply involved in the process of infection of the host. This also suggests that the second phage is significantly different in morphology and properties from Xp12, and that this difference leads to a difference in host range.
  • the search range was further expanded to search for sequences with 80% or more sequence identity (Identity) over 80% or more of the total length (Query Cover) to the genomic DNA sequence of the second phage.
  • sequences with 80% or more sequence identity (Identity) over 80% or more of the total length (Query Cover) to the genomic DNA sequence of the second phage.
  • Example 3 Isolation of a novel bacteriophage and its lytic activity (3) (the purpose) We will isolate novel bacteriophages that have lytic activity against pathogenic bacteria that cause plant diseases, and verify their lytic activity against plant pathogenic bacteria.
  • NCIMB-ID a strain of Pseudomonas fluorescens, a bacterium of the genus Pseudomonas, was obtained from NCIMB, a research institute within the UK National Collection of Microorganisms (UKNCC) (NCIMB-ID: 10460).
  • Xanthomonas bacteria and Pseudomonas fluorescens were cultured according to the method described in "(1) Obtaining and culturing plant pathogenic bacteria" in Example 1.
  • the isolated phages were suspended in SM Buffer and passed through a 0.2 ⁇ m filter to recover the phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to purify the phages.
  • a plate lysate (PL) method was carried out to amplify and purify the isolated and purified third phage.
  • the specific method was in accordance with the method described in "(3) Amplification and purification of phage" in Example 1.
  • the titer of the prepared purified third phage solution was determined by a plaque assay method using an appropriately diluted solution, and was confirmed to be 10 8 PFU/mL or more.
  • the third phage may be useful in controlling diseases caused by Xanthomonas bacteria, such as walnut bacterial black spot, tomato bacterial spot, lettuce bacterial spot, and broccoli black rot.
  • sequence identity over the full length corresponds to a value of about 85.20%.
  • known phage with the second highest genome DNA sequence identity (phage name: BUCT598, access code: MW831865.1, sequence identity over the full length: about 85.18%)
  • the known phage with the third highest genome DNA sequence identity (phage names: BUCT703 and BUCT700, access codes: OM735688.1 and OM735686.1, respectively, sequence identity over the full length: both about 56.7%) both targeted bacteria of the genus Stenotrophomonas.
  • Example 4 Lytic activity of the obtained bacteriophage combination (1) (the purpose) We will verify the effectiveness of new bacteriophages that have been shown to have lytic activity when used alone against pathogenic bacteria that cause plant diseases, when used in combination with other bacteriophages, against plant diseases.
  • the phage purified solution to be dripped was a mixed solution prepared by mixing equal amounts of the phage purified solutions prepared in each Example.
  • the mixed solution was appropriately diluted so that the total titer was equivalent to that when each solution was used alone.
  • the phages used in this Example are as follows.
  • a first phage having a genomic DNA sequence of SEQ ID NO:1 (a in FIG. 6B) was used in combination with a phage having a genomic DNA sequence of SEQ ID NO:4 (b in FIG. 6B), a phage having a genomic DNA sequence of SEQ ID NO:5 (c in FIG. 6B), a phage having a genomic DNA sequence of SEQ ID NO:6 (d in FIG. 6B), a phage having a genomic DNA sequence of SEQ ID NO:7 (e in FIG. 6B), a second phage having a genomic DNA sequence of SEQ ID NO:2 (f in FIG. 6B), a third phage having a genomic DNA sequence of SEQ ID NO:3 (g in FIG. 6B), and a phage having a genomic DNA sequence of SEQ ID NO:8 (h in FIG. 6B).
  • Example 5 Lytic activity of the obtained bacteriophage combination (2) (the purpose) We will verify the effectiveness of new bacteriophages that have been shown to have lytic activity when used alone against pathogenic bacteria that cause plant diseases, when used in combination with other bacteriophages, against plant diseases.
  • the phage purified solution to be dripped was a mixed solution prepared by mixing equal amounts of the phage purified solutions prepared in each Example.
  • the mixed solution was appropriately diluted so that the total titer was equivalent to that when each solution was used alone.
  • the phages used in this Example are as follows.
  • a second phage having a genomic DNA sequence of SEQ ID NO:2 (a in FIG. 7B) was used in combination with a phage having a genomic DNA sequence of SEQ ID NO:4 (b in FIG. 7B), a phage having a genomic DNA sequence of SEQ ID NO:5 (c in FIG. 7B), a phage having a genomic DNA sequence of SEQ ID NO:6 (d in FIG. 7B), a phage having a genomic DNA sequence of SEQ ID NO:7 (e in FIG. 7B), a first phage having a genomic DNA sequence of SEQ ID NO:1 (f in FIG. 7B), a third phage having a genomic DNA sequence of SEQ ID NO:3 (g in FIG. 7B), and a phage having a genomic DNA sequence of SEQ ID NO:8 (h in FIG. 7B).
  • Example 6 Lytic activity of the obtained bacteriophage combination (3) (the purpose) We will verify the effectiveness of new bacteriophages that have been shown to have lytic activity when used alone against pathogenic bacteria that cause plant diseases, when used in combination with other bacteriophages, against plant diseases.
  • the phage purified solution to be dripped was a mixed solution prepared by mixing equal amounts of the phage purified solutions prepared in each Example.
  • the mixed solution was appropriately diluted so that the total titer was equivalent to that when each solution was used alone.
  • the phages used in this Example are as follows.
  • a third phage having a genomic DNA sequence of SEQ ID NO:3 (a in FIG. 8B) was used in combination with a phage having a genomic DNA sequence of SEQ ID NO:4 (b in FIG. 8B), a phage having a genomic DNA sequence of SEQ ID NO:5 (c in FIG. 8B), a phage having a genomic DNA sequence of SEQ ID NO:6 (d in FIG. 8B), a phage having a genomic DNA sequence of SEQ ID NO:7 (e in FIG. 8B), a first phage having a genomic DNA sequence of SEQ ID NO:1 (f in FIG. 8B), a second phage having a genomic DNA sequence of SEQ ID NO:2 (g in FIG. 8B), and a phage having a genomic DNA sequence of SEQ ID NO:8 (h in FIG. 8B).
  • Example 7 Tomato bacterial spot disease control effect test (the purpose) We will verify the effectiveness of isolated novel bacteriophages that have lytic activity against pathogenic bacteria of plant diseases when applied to plants.
  • phage spray solution used in this test was prepared by the following procedure.
  • a host bacterial cell culture for phage amplification was prepared as follows.
  • the host used was Xanthomonas campestris pv. vesicatoria (MAFF No. 301256), a strain confirmed to have bacteriolytic activity in all phages tested.
  • the bacterial cells were inoculated into YPG Broth and incubated overnight in a shaker set at 25°C. After incubation, OD 600 (turbidity at a wavelength of 600 nm) was measured, and those that reached approximately 1.0 were used as the bacterial cell culture below.
  • the prepared bacterial culture solution and the phage purification solution (titer of about 10 8 PFU/mL) containing one type of phage prepared in Example 1 were mixed in equal amounts and inoculated into 100 times the amount of YPG culture solution, and the resulting culture solution was recovered as a phage crude solution after incubating for about 8 to 12 hours in a shaker set at 25°C. 1/10 the amount of chloroform was added to the recovered crude solution, and after vigorously stirring, the solution was centrifuged at 8,000g/20°C/5 minutes to recover the supernatant. The recovered supernatant was passed through a 0.2 ⁇ m filter, and the filtrate was used as a phage purification solution.
  • a phage purified solution was diluted with sterilized tap water to a titer of about 10 9 PFU/mL and used.
  • the bacterial spray solution used for infecting plants in this test was prepared using the bacterial cell culture solution prepared in (1).
  • the bacterial cell culture solution diluted approximately 10,000-fold with sterile tap water was applied to a YPG agar plate and incubated in an incubator set at 25°C for approximately 1-3 days.
  • the bacterial cell suspension recovered by suspending colonies on the YPG agar plate in sterile tap water was diluted with sterile tap water to a final OD 600 of approximately 0.5 to prepare the bacterial spray solution.
  • the phage solution was sprayed twice on the leaves of each specimen, twice before and twice after the bacterial infection treatment, with an interval of 2-3 days, and then the bacterial solution was sprayed on the leaves 2-3 days later to infect the specimens with bacteria.
  • the infected specimens were left to stand in a vinyl greenhouse with high humidity for about 2 days after the infection treatment.
  • the phage solution was then sprayed on the leaves again twice with an interval of 2-3 days.
  • the non-treated group the treatment was carried out in the same manner as above, except that sterilized tap water was used instead of the phage purified solution.
  • Leaves showing the characteristics of tomato bacterial spot disease on their surface were judged to be diseased, and the ratio of diseased leaves to the total number of leaves was calculated as the disease incidence rate.
  • the relative disease incidence rate in the phage-treated group was calculated as a relative value when the disease incidence rate in the untreated group was set at 100%.
  • the phages of the present invention can effectively protect plants from disease even when applied to actual plants.
  • Example 8 Broccoli black rot disease control effect test (the purpose) We will verify the effectiveness of isolated novel bacteriophages that have lytic activity against pathogenic bacteria of plant diseases when applied to plants.
  • phage spray solution A phage spray solution was prepared in the same manner as in Example 7, except that the bacterium used as the bacterial cell was Xanthomonas campestris pv. campestris (MAFF No. 106765) and the following phages were used.
  • the phage spray solution containing multiple types of phages was prepared by mixing equal amounts of purified phage solutions, each of which had been adjusted to have the same order of titer, and diluting the mixture with sterile tap water to a titer of approximately 10 9 PFU/mL.
  • a third phage having a genomic DNA sequence of SEQ ID NO:3 was used in combination with a phage having a genomic DNA sequence of SEQ ID NO:9 (b in FIG. 10), or with an additional phage having a genomic DNA sequence of SEQ ID NO:8 (c in FIG. 10) and a phage having a genomic DNA sequence of SEQ ID NO:10 (d in FIG. 10), or with an additional phage having a genomic DNA sequence of SEQ ID NO:5 (e in FIG. 10) and a phage having a genomic DNA sequence of SEQ ID NO:6 (f in FIG. 10) in combination with a phage having a genomic DNA sequence of SEQ ID NO:7 (g in FIG. 10) and a phage having a genomic DNA sequence of SEQ ID NO:11 (h in FIG. 10).
  • Example 7 Measurement of Disease Incidence The same procedure as in Example 7 was followed, except that the evaluation was carried out 16 days after infection.
  • the relative disease incidence rate further decreased to about 45.7% on average.
  • the relative disease incidence rate was about 60% ("a/b” in Figure 10), but generally, the more types combined, the higher the disease control effect and the lower the relative disease incidence rate tended to be.
  • the relative disease incidence rate was about 43.5% ("a/b/c/d” in Figure 10)
  • the relative disease incidence rate was about 33.5% ("a/b/c/d/e/f/g/h” in Figure 10).
  • the phages of the present invention can effectively protect plants from disease, even when applied to actual plants, regardless of the type of plant itself. It was also found that a higher effect can be obtained by applying the phages of the present invention in combination.

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