Bacteriophage, plant disease control agent containing bacteriophage, and method for producing bacteriophage

Abstract

To provide a novel bacteriophage capable of pest-controlling a rice seedling disease and a bacterial plant disease such as ralstonia.SOLUTION: The bacteriophage found in organic cultivation nursery soil contains a nucleic acid molecule including a novel base sequence or a nucleic acid molecule including a base sequence having homology of 95% or more to the novel base sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a bacteriophage, a plant disease control agent containing the bacteriophage, and a method for producing the bacteriophage. [Background technology]

[0002] Plant diseases are a cause of losses during the production process of agricultural crops, and bacterial plant diseases such as rice seedling disease and bacterial wilt are becoming a problem. For example, the rice seedling rot fungus Burkholderia glumae and the rice seedling blight fungus B. plantarii are pathogenic bacteria that cause rice seedling rot and are therefore major problems during the seedling raising process of paddy rice. Bacterial wilt is a plant disease caused by the bacterial wilt fungus Rastonia pseudosolanacearum, which infects and kills more than 200 species of plants, including those of the Solanaceae family, and the damage caused by bacterial wilt is serious in agriculture.

[0003] The mainstream method of controlling these pests is to use chemical pesticides, but due to problems such as the emergence of resistant bacteria and environmental burden, the development of new control methods is required. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Organic Agriculture volume 4, pages 187-196(2014) [Non-Patent Document 2] Jouenal of Phytophathology Volume 166, Issue 4 April 2018, p249-264 Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention is to provide a novel bacteriophage capable of controlling bacterial plant diseases such as rice seedling diseases and bacterial wilt. [Means for solving the problem]

[0006] Under such circumstances, the present inventors have suggested that organic seedling soil has an inhibitory effect against rice bacterial grain rot (Non-Patent Document 1), and that this effect may be related to a diverse and stable bacterial community (Non-Patent Document 2). However, the involvement of phages in the disease inhibitory effect of organic seedling soil has not been examined. Therefore, the present inventors have attempted to isolate phages from organic seedling soil, and after a great deal of trial and error, have found that the above-mentioned problems can be solved by a bacteriophage obtained by a method including the steps of: inducing a bacteriophage group including a target bacteriophage from organic seedling soil; culturing the phage group in the presence of host cells to grow the target phage; removing the host cells from the culture solution; culturing the culture solution from which the host cells have been removed using a soft agar medium to which the host cells have been added, and forming plaques of the phage that infect the host cells. The present invention is based on such novel findings. Thus, typically, the present invention provides the following:

[0007] Item 1. A bacteriophage comprising a nucleic acid molecule comprising a base sequence represented by SEQ ID NO:1, or a nucleic acid molecule comprising a base sequence having 95% or more homology to the base sequence represented by SEQ ID NO:1.

[0008] Item 2. The bacteriophage according to Item 1, which is a jumbo phage.

[0009] Item 3. A plant disease control agent comprising the bacteriophage according to item 1 or 2.

[0010] Item 4. The plant disease control agent according to Item 3, wherein the plant disease is one or both of a rice seedling disease and bacterial wilt disease.

[0011] Item 5. A process for extracting a bacteriophage population including a target bacteriophage from organic soil Cultivating the phage population in the presence of host cells to grow the phages of interest. Removing host cells from the culture medium A step of culturing the culture solution from which the host cells have been removed in a soft agar medium to which the host cells have been added, to form phage plaques that infect the host cells. A method for producing a bacteriophage that infects a host cell, comprising:

[0012] Item 6. Mixing organic soil with liquid medium A step of centrifuging and / or filtering the mixture of organic soil and liquid medium obtained in the above step. adding host cells to the supernatant or filtrate obtained in the above step and culturing them; A step of centrifuging and / or filtering the culture solution obtained in the above step. A step of culturing the supernatant or filtrate obtained in the above step using a soft agar medium containing host cells. A method for producing a bacteriophage that infects a host cell, comprising:

[0013] Item 7. The method according to Item 5 or 6, wherein the host cell is at least one species selected from the group consisting of bacteria of the genus Burkholderia and bacteria of the genus Ralstonia.

[0014] Item 8. The method according to any one of Items 5 to 7, for obtaining the bacteriophage according to Item 1 or 2. Effect of the Invention

[0015] According to the present invention, a novel bacteriophage capable of controlling bacterial plant diseases such as rice seedling diseases and bacterial wilt can be provided. [Brief description of the drawings]

[0016] [Figure 1]Morphology of FLC6 particles and plaques. (A) FLC6 formed clear plaques in B. glumae MAFF302552. (B) FLC6 particles with contractile tails that were not contracted and (C) FLC6 particles with contractile tails were observed using a transmission electron microscope. [Diagram 2] Comparison of the full-length genome sequences of Ralstonia phage RSL2 (RefSeq accession number NC_028950.1) and Ralstonia phage RSF1 (RefSeq accession number NC_028899.1), which have sequence similarity to FLC6. Arrows indicate ORFs. Grey bands indicate nucleotide sequence homology. [Figure 3A] Phylogenetic tree of FLC6 and other phages based on their sequences. (A) Proteomic tree of FLC6 and other phages, with Escherichia virus P2 used as outgroup. [Figure 3B] (B) Phylogenetic tree based on the amino acid sequences of the putative major head protein (gp125) of FLC6 and homologous proteins of other phages. Genus names were taken from the ICTV #35 master species list. [Figure 4] Suppression of rice seedling rot by FLC6 treatment. (A) Percentage of rice seedling rot symptoms in B. glumae MAFF302746-contaminated seeds immersed in FLC6 and in the control area. The control area was immersed in PPG liquid medium diluted to the same concentration as the phage dilution solution. (B) Phage-treated plants (8 days after inoculation) of B. glumae MAFF302746-contaminated seeds and healthy seeds. [Diagram 5] Overview of the method for producing the bacteriophage of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] In this specification, "nucleic acid" is synonymous with nucleotide, oligonucleotide, and polynucleotide, and may be any of DNA, RNA, and DNA-RNA hybrids. These may be double-stranded or single-stranded, and when a nucleic acid molecule having a certain sequence is mentioned, it is meant to comprehensively mean a nucleic acid molecule (or nucleotide, oligonucleotide, and polynucleotide) having a complementary sequence, unless otherwise specified. These nucleic acid molecules may be circular or linear, and may be synthetic or of biological origin.

[0018] Bacteriophage In a typical embodiment, the present invention provides a bacteriophage containing a nucleic acid molecule comprising the base sequence shown in SEQ ID NO: 1. Furthermore, the bacteriophage according to the present invention is not limited to the above as long as it exhibits the effects of the present invention, and in a preferred embodiment of the present invention, the present invention provides a bacteriophage containing a nucleic acid molecule comprising a base sequence having 95% or more homology to the base sequence shown in SEQ ID NO: 1.

[0019] In the present invention, the homology of base sequences can be calculated using analytical software that is commercially available or available via the Internet, for example, the BLAST program of NCBI.

[0020] In this embodiment, the nucleic acid molecule possessed by the bacteriophage of the present invention preferably has a homology of 95% or more, more preferably 97% or more, more preferably 99% or more, and even more preferably 99.9% or more to the base sequence represented by SEQ ID NO: 1. A typical example of a nucleic acid molecule possessed by the bacteriophage of the present invention and containing the base sequence represented by SEQ ID NO: 1 or a base sequence having 95% or more homology to the base sequence represented by SEQ ID NO: 1 is a DNA molecule. More specifically, in a typical embodiment, the bacteriophage of the present invention has the above-mentioned nucleic acid molecule as genomic DNA.

[0021] The bacteriophage of the present invention is useful because it not only has a control effect against rice seedling diseases, but also has a control effect against bacterial wilt. Generally, a bacteriophage that is effective against rice seedling damping-off is not necessarily effective against bacterial wilt caused by a completely different bacterium. Therefore, the effect of the bacteriophage of the present invention that can control both of these diseases cannot be easily predicted.

[0022] In a typical embodiment, the bacteriophage of the present invention is a jumbo phage. In the present invention, the jumbo phage means a bacteriophage having a genome size of 200,000 bp or more. The bacteriophage of the present invention can be produced, for example, by the method described in the "Method for producing a bacteriophage" below.

[0023] Plant disease control agents In another embodiment, the present invention provides a plant disease control agent comprising a bacteriophage.

[0024] As used herein, "control" refers to prevention, eradication, or both. Plant disease includes preventing plant disease and suppressing or eliminating plant disease.

[0025] Plants whose diseases are controlled by the bacterial population of the present invention are not particularly limited, and may be vegetables, potatoes, mushrooms, fruits, beans, grains, nuts, seeds, ornamental plants, ferns, mosses, etc., with grains and vegetables being preferred. Grains include rice, wheat, barley, oats, rye, foxtail millet, millet, barnyard millet, corn, etc., with rice being preferred. Vegetables include solanaceae vegetables such as tomatoes, eggplants, bell peppers, shishito peppers, paprika, and chili peppers; cruciferous vegetables such as Chinese cabbage, cabbage, and broccoli; and Cucurbitaceae vegetables such as cucumbers and pumpkins, with solanaceae plants such as tomatoes, eggplants, bell peppers, shishito peppers, paprika, and chili peppers being preferred.

[0026] The plant disease control agent according to the present invention is particularly effective against various pathogenic bacteria that cause diseases in Gramineae plants, Solanaceae plants, etc. Examples of pathogenic bacteria that cause diseases in Gramineae plants include the genus Burkholderia (e.g., Burkholderia glumae), which is a bacterial rice grain blight pathogen, the genus Psudomonas (e.g., Psudomonas avenae), which is a bacterial rice brown stripe pathogen, the genus Burkholderia (e.g., Burkholderia plantarii), which is a bacterial rice seedling blight pathogen, and the genus Xanthomonas (e.g., Xanthomonas oryzae pv. oryzae), which is a bacterial rice leaf blight pathogen. Examples of pathogenic fungi that cause diseases in solanaceae plants include the genus Ralstonia (e.g., Ralstonia pseudosolanacearum, R. syzygii subsp. indonesiensis), which causes bacterial wilt, and the genus Erwinia (e.g., Erwinia carotovora subsp. carotovora), which causes soft rot. Other pathogenic fungi include the genus Xanthomonas (e.g., Xanthomonas campestris pv. campestris), which causes black rot.

[0027] The concentration of the bacteriophage in the plant disease control agent of the present invention is not particularly limited and can be appropriately set depending on the type and severity of the disease to be treated, the application method, and the formulation type. 4 CFU / mL or more is preferable, and 1×10 5 ~10 13 It is preferable to set the range to CFU / mL.

[0028] The plant disease control agent of the present invention can be used as a bacteriophage culture as is or after dilution, or a suspension can be prepared by diluting the bacteriophage in a solvent such as water, a buffer solution, etc. The plant disease control agent of the present invention can also be prepared as various formulations such as wettable powders, wettable granules, and emulsifiable concentrates, in the same manner as known plant disease control agents.

[0029] By treating plant seeds, seedlings, seedling soil or seedling medium with the plant disease control agent of the present invention, plant diseases can be controlled.

[0030] Treatment of plant seeds, seedlings, seedling soil or seedling medium with a plant disease control agent can be carried out by a method appropriate for the type of disease, the degree of infection, the application range, the formulation type, etc. Examples of such treatments include seed soaking treatment, seed dressing treatment, seed coating treatment, seed spraying treatment, incorporation into cover soil, incorporation into bed soil, application to seedling boxes, liquid irrigation, base application, side stripe application, water surface application, spraying by unmanned aerial vehicle, etc.

[0031] In addition, when applying the plant disease control agent of the present invention, it may be mixed with an insecticide, a nematicide, acaricide, herbicide, plant growth promoter, fertilizer, soil improvement material, etc., or may be used in combination with them alternately or simultaneously without mixing.

[0032] Method for producing bacteriophage In a preferred embodiment, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: A process for extracting a bacteriophage population including a target bacteriophage from organic soil. Cultivating the phage population in the presence of host cells to grow the phages of interest. Removing host cells from the culture medium A step of culturing the culture solution from which the host cells have been removed in a soft agar medium to which the host cells have been added, to form phage plaques that infect the host cells. A method for producing a bacteriophage that infects a host cell, comprising: to provide.

[0033] In the method of the present invention, a step of extracting a bacteriophage group including a target bacteriophage from organic soil is first performed. In the present invention, the "target bacteriophage" typically refers to a bacteriophage that can infect a microorganism causing a plant disease and control the plant disease caused by the microorganism.

[0034] In the present invention, the term "organic soil" refers to soil containing organic matter used as fertilizer. Examples of organic soil include leaf compost, bark, oil cake, mushroom cultivation residue, sawdust, moss, charcoal, vegetation charcoal, peat, peat moss, leaf mold, rice bran, rice straw, rice husk, bran, tree fiber, materials derived from fermented, dried or burned animal excrement, materials from food factories or textile factories, materials derived from food waste, compost containing one or more selected from these, and seedling soil containing one or more selected from these. Organic soil is not heat-treated for sterilization before plant seedlings are raised. Heat treatment usually refers to treatment at 70°C or higher for 30 minutes or more.

[0035] The elicitation step is carried out, for example, by mixing the organic soil with a liquid medium. The liquid medium is not particularly limited as long as it can be used for culturing bacteriophage, and examples thereof include Potato-Peptone-Glucose (PPG) liquid medium, Lysogeny Brothe (LB) liquid medium, Nutrient Broth (NB) liquid medium, Casamino acid-Peptone-Glucose (CPG) liquid medium, etc., and includes PPG medium. In addition, the medium is preferably one that is also suitable for culturing the host cells added in the subsequent step. The PPG liquid medium may be used after dilution, and the dilution ratio when diluting may be, for example, 1000 times or less. For example, the composition of PPG liquid medium is a mixture of 0.5% (w / v) peptone, 0.5% (w / v) glucose, 0.3% (w / v) Na2HPO4·12H2O, and 0.05% (w / v) KH2PO4 in a decoction made by adding 200 g of potatoes to 1 L of distilled water and boiling the mixture. The composition of LB liquid medium is 5 g of yeast extract, 10 g of tryptone, and 5 g of NaCl per 1 L of medium. The composition of NB liquid medium is 30 g of dried bouillon per 1 L of medium. The composition of CPG liquid medium is 10 g of peptone, 1 g of casamino acid, and 5 g of glucose per 1 L of medium. The above-mentioned medium may contain carbon sources such as glucose and starch; pH adjusters such as phosphoric acid or its salts; antifoaming agents such as animal oil, vegetable oil, and mineral oil; and metal salts. The blending ratio of organic soil and liquid medium is not particularly limited, and for example, the liquid medium can be appropriately set within a range of 10 to 500 parts by mass, preferably 30 to 100, per 10 parts by mass of organic soil. The temperature during mixing is not particularly limited, and can be appropriately set within a range of 16 to 45° C., more preferably 20 to 40° C. The mixing time is also not particularly limited, and can be appropriately set within a range of, for example, 10 minutes to 3 hours, more preferably 30 minutes to 2 hours.

[0036] The mixture of organic soil and liquid medium obtained in the induction step may be used as is in the next phage propagation step, but it is preferable to centrifuge and / or filter the mixture to remove soil particles, etc., and use the supernatant in the next step. The centrifugation and filtration methods are not particularly limited as long as they can remove soil particles, etc., while leaving the target bacteriophage in the supernatant or filtrate, and any method commonly used in the field to which the present invention pertains can be used.

[0037] Next, a step of culturing the phage population in the presence of host cells to grow the phages of interest is carried out. This step is carried out by adding host cells to the mixture, supernatant or filtrate obtained in the above step and culturing the cells. In a typical embodiment of the present invention, a microorganism that causes plant diseases is used as the host cell. The amount of the host cells to be added at the time of addition is not particularly limited, but may be, for example, 10 5 ~10 9cfu / mL, preferably 10 6 ~10 8 The range of the mixing temperature is preferably cfu / mL. The culture may be static culture or shaking culture, but shaking culture is preferred. The temperature during culture is not particularly limited, and can be appropriately set within the range of 16 to 45°C, more preferably 20 to 40°C. The mixing time is also not particularly limited, and can be appropriately set, for example, within the range of 8 to 30 hours, more preferably 10 to 24 hours.

[0038] Next, a step of removing host cells from the culture solution is performed. This step can be performed, for example, by centrifuging and / or filtering the culture solution obtained in the above step. By once removing the host cells from the medium in this step, phages capable of infecting the host cells can be selected in the next plaque formation step. The conditions for centrifugation are not particularly limited as long as the host cells can be removed by the centrifugation alone or by combining the centrifugation and the filtration step, and the target bacteriophage can be left behind, and can be appropriately set, for example, within the range of 3000 to 12000 g, preferably 4000 to 6000 g. The centrifugation time is also not particularly limited, and can be appropriately set, for example, within the range of 1 to 15 minutes, preferably 3 to 10 minutes. The filter used for filtration is not particularly limited as long as the host cells can be removed by the filtration alone or by combining the filtration step and centrifugation, and the target bacteriophage can be left behind, and can be appropriately set, for example, with a pore size of 0.2 to 1 μm, preferably 0.3 to 0.5 μm. The filtration may be performed multiple times using filters with different pore sizes (e.g., a 0.3-0.5 μm filter and a 0.7-0.9 μm filter, etc.). The temperature during the centrifugation and filtration is not particularly limited and can be appropriately set within the range of 20 to 40° C., more preferably 20 to 30° C. In this step, it is preferable to perform both centrifugation and filtration from the viewpoint of removing host cells.

[0039] Next, the culture solution from which the host cells have been removed is cultured using a soft agar medium to which the host cells have been added, and a step of forming plaques of phages that infect the host cells is performed. This step can be performed, for example, by culturing the supernatant or filtrate obtained in the above step using a soft agar medium to which the host cells have been added. In the present invention, the soft agar medium means an agar medium having an agar concentration of 0.8% or less. As the soft agar medium, a liquid medium such as PPG liquid medium, LB medium, NB medium, CPG medium, etc. to which agar has been added can be used. The amount of agar to be added can be appropriately set, for example, within the range of 0.3 to 0.8% by mass, preferably 0.35 to 0.5% by mass, as the final concentration of agar in the medium when plaques are formed. Plaques of phages with a slow diffusion rate, such as jumbo phages, can also be detected. As the host cells, those described above can be used. According to the present invention, by using a soft agar medium, the amount of host cells to be added at the time of addition is not particularly limited, but for example, 10 5 ~10 9 cfu / mL, preferably 10 6 ~10 8 The range of the concentration of the culture medium is preferably in the range of cfu / mL. The temperature during the culture is not particularly limited and can be appropriately set in the range of 16 to 45°C, more preferably in the range of 20 to 40°C. The mixing time is also not particularly limited and can be appropriately set, for example, in the range of 8 to 30 hours, more preferably in the range of 10 to 24 hours. In the present invention, the step can be performed by a double plate method in which a soft agar medium containing the supernatant or filtrate and the host cells is layered on a plate medium and cultured. The target bacteriophage can be obtained from the plaques formed in the step.

[0040] According to the present invention, the bacteriophage of interest can be efficiently obtained by carrying out each of the above steps, in particular by using organic soil as a source for isolating the bacteriophage of interest, adding host bacteria for amplification for isolation, and using soft agar medium to detect phage plaques.

[0041] The following examples are provided to further illustrate the present invention, but the present invention is not limited to these examples. EXAMPLES

[0042] method Bacterial strains and culture conditions In this study, we used the following bacterial strains: Burkholderia gulmae (MAFF nos. 302552, 106619, 301169, 302417, 302746), B. plantarii (106726, 106727, 301723, 302475, 302909, 302936), Ralstonia pseudosolanacearum (106603, 106611, 211270, 301485), and R. syzygii subsp. indonesiensis (211271, 327032). All strains were obtained from the National Agriculture and Food Research Organization (NARO) Genebank.

[0043] Burkholderia bacteria were cultured in Potato-Peptone-Glucose (PPG) medium (0.5% (w / v) peptone, 0.5% (w / v) glucose, 0.3% (w / v) Na2HPO4 12H2O, and 0.05% (w / v) KH2PO4 were mixed with a decoction made by adding 1 L of distilled water to 200 g of potatoes), and Ralstonia bacteria were cultured in Casamino acid-Peptone-Glucose (CPG) medium (1.0% (w / v) peptone, 0.1% (w / v) casamino acid, and 0.5% (w / v) glucose). 1.5% (w / v) agar plates were used for culture of bacteria from stocks, and 0.5% (w / v) agar soft agar plates were used for double plating.

[0044] B. glumae and B. plantarii were suspended in skim milk dispersion medium (1% (w / v) sodium glutamate, 10% (w / v) skim milk) and stored at -80°C. Cultures from stocks were grown on plates at 25°C for 2 days. R. solanacearum and R. syzygii subsp. indonesiensis were suspended in 20% (v / v) glycerol and stored at -80°C. Cultures from stocks were grown on plates at 25°C for 2 days. Bacterial cells on the plates were suspended in sterile water to prepare bacterial suspensions.

[0045] Phage isolation and culture conditions The leaf compost used in the phage search was made in an experimental field at the Graduate School of Agricultural Science, Tohoku University. 135 L of broadleaf leaves and 45 L of rice husks were layered together, and 20 L of water was added. The temperature of the compost was continuously measured, and when it exceeded 60°C and became isothermal with the air temperature, it was turned over. This procedure was repeated five times to maintain the aerobic conditions necessary for the compost to mature.

[0046] Leaf compost (fresh weight 6.5 g) was suspended in 25 mL of PPG liquid medium and vigorously shaken at room temperature for 2 hours. The mixture was centrifuged at 5000 xg for 5 minutes at room temperature, and the supernatant was filtered through 0.8 μm and 0.45 μm pore-sized mixed cellulose ester filters in sequence. Approximately 1 mL of B. glumae MAFF302552 suspension (final concentration 10 7 cfu / mL) and CaCl2 (final concentration 1 mM) were added and incubated overnight at 180 rpm at 25°C with a rotary gyroscope. The mixture was centrifuged at 5000 xg for 5 minutes at room temperature, and the supernatant was filtered through 0.8 μm and 0.45 μm pore size filters (cellulose mixed esters). Plaques (clear areas where bacteria do not grow) were detected from the filtrate by the double plate method. In the double plate method, 0.5 mL of the filtrate and a B. glumae MAFF302552 suspension (10 8cfu / mL) was mixed and left to stand at 25°C for 10 minutes, after which 5 mL of soft agar medium was added and mixed, and the mixture was layered on the plate medium (final agar concentration: 0.42%). The mixture was cultured overnight at 25°C, and the presence or absence of plaques was observed.

[0047] Plaques were excised together with the surrounding host bacteria and suspended in phage buffer (10 mM Tris-HCl, pH 7.5, 68.5 mM NaCl, 10 mM MgSO4 and 1 mM CaCl2). Serial dilutions were performed and sparse plaques were formed again by double plating. Single plaques were obtained three times and phages were isolated. The obtained phages were amplified by rotating culture at 180 rpm and 25°C overnight with B. glumae MAFF302552 in PPG liquid medium, then centrifuged at 5000 xg for 5 minutes at room temperature, and the supernatant was filtered sequentially through 0.8 μm and 0.45 μm pore size filters (cellulose mixed esters) to remove bacteria. The phage amplified solution was stored at 4°C.

[0048] Host range analysis was performed by spot testing. 8 cfu / mL) was mixed with 5 mL of soft agar medium and layered on a plate. After the soft agar medium solidified, 10 μL of the phage amplification solution and 10 μL of the phage buffer as a control were dropped onto the plate, and the plate was incubated overnight at 25°C to confirm the formation of plaques.

[0049] Electron microscope observation The phage amplified solution was ultracentrifuged at 45,000 rpm for 90 minutes at 4°C to obtain a phage concentrate. The rotor S50A-2521 and ultracentrifuge himac CS100FNX (Koki Holdings) were used for ultracentrifugation. The phage concentrate was dropped onto a 400-mesh collodion membrane-attached mesh (Nissin EM), stained with phosphotungstic acid, and observed under a transmission electron microscope H-7650 (Hitachi High-Tech).

[0050] DNA extraction and next-generation sequencing Genomic DNA of FLC6 was extracted from the concentrated phage solution by phenol-chloroform extraction according to the standard method. Libraries were prepared using the Nextera XT kit v2 (Illumina), and 2 x 150 bp sequences were sequenced on a MiSeq (Illumina) using the MiSeq v2 Reagent Kit Nano (Illumina).

[0051] In silico analysis Next-generation sequencing reads were assembled using SPAdes v3.11.1. Homology searches of the full-length genome sequence of FLC6 were performed using BLASTn against the NCBI Nucleotide collection. ORFs were identified using GeneMarkS v4.28. tRNA and rRNA sequences were searched using tRNAscan-SE v2.0 and RNAmmer 1.2, respectively.

[0052] The full-length genome sequences and ORFs of FLC6, Ralstonia phage RSL2, and Ralstonia phage RSF1 were illustrated using EasyFig. A molecular phylogenetic tree was constructed between the putative major head protein of FLC6 and proteins with homologous sequences of other phages based on amino acid sequences. The phylogenetic tree was constructed using the NJ method with clustalW v2.1 on the DDBJ website. A proteomic tree based on tBLASTx was constructed using ViPTree v1.9.

[0053] Evaluation of disease suppression effect against rice seedling rot The rice 'Koshihikari' was used for the bioassay. B. glumae MAFF302746 suspension (10 7Rice seeds were immersed in 100 ml of 1000 cfu / mL of phage amplicons and inoculated under reduced pressure for 5 minutes to prepare contaminated seeds. After vacuum inoculation, the seeds were air-dried overnight and stored at 4°C until use. The phage amplification solution was diluted with water and diluted to 10 7 A phage solution of pfu / mL was prepared, and the contaminated seeds and healthy seeds before inoculation were soaked in the phage solution for 1 day at 28°C. As a control, the seeds were soaked in a diluted PPG liquid medium prepared by mixing the phage solution and medium components in equal concentrations. The soaking solution was discarded, and the seeds were rinsed three times with tap water, and soaked in tap water for 1 day at 28°C.

[0054] Germinated seeds were sown in autoclaved commercial nursery soil L (Sanken Soil). 7.5 x 7.5 x 5.5 cm plastic containers were used, with 100 g of bed soil and 50 g of cover soil per pot, and the water level was set to cover the surface. The plants were cultivated in an incubator at 30°C, 14L10D for 8 days, and the disease score of each individual was evaluated on a four-point scale: 0: no symptoms, 1: growth inhibition / yellowing, 2: half dead, 3: complete dead. A U-test was performed on the number of individuals per disease score between the contaminated seeds-phage treatment plot and the contaminated seeds-control plot. The disease severity for each treatment plot was calculated using the following formula. [(1A + 2B + 3C) / 3N] × 100 (A: number of individuals with disease score 1, B: number of individuals with disease score 2, C: number of individuals with disease score 3, N: total number of individuals). The control titer was calculated as 100 - (disease severity in FLC-treated areas / disease severity in control areas) × 100.

[0055] result Isolation from leaf compost and observation of particle structure Plaque detection experiments in leaf compost confirmed the presence of transparent plaques approximately 0.5 mm in diameter (Fig. 1A). A single plaque was isolated and designated FLC6. Observation of the phage particles using a transmission electron microscope revealed a head-and-tail type particle structure with a contractile tail (Fig. 1B, C), suggesting that the phage belongs to the Myoviridae family.

[0056] Genomic sequence determination and analysis of FLC6 Since phages of the Myoviridae family are known to have double-stranded DNA in their genomes, genomic DNA was extracted from FLC6 particles and sequenced using a next-generation sequencer. As a result, it was revealed that FLC6 has a genome sequence of 227,105 bp. The FLC6 genome DNA sequence is shown in SEQ ID NO:1. Phages with a genome size of more than 200,000 bp are called jumbo phages. There have been no reports of jumbo phages that lyse plant pathogenic bacteria of the genus Burkholderia. 241 ORFs were identified in the FLC6 genome. No tRNA or rRNA sequences were found in the FLC6 genome.

[0057] A BLASTn search using the full-length genome sequence of FLC6 as a query and the NCBI Nucleotide collection as a database confirmed homology with two previously reported jumbo phages that infect Ralstonia solanacearum, Ralstonia phage RSL2 (88% query coverage, 90% identity) and Ralstonia phage RSF1 (79% and 85% identity) (Fig. 2). In addition, based on the amino acid sequence of the major head protein, a molecular phylogenetic tree constructed by the NJ method using clustalW v2.1 (Fig. 3A) and a proteomic tree constructed by ViPTree, a program using tBLASTx (Fig. 3B), FLC6 was included in the same clade as Ralstonia phage RSL2 and RSF1. Since both Ralstonia phage RSL2 and RSF1 belong to the Chiangmaivirus genus, FLC6 is also thought to belong to the Chiangmaivirus genus.

[0058] Host range analysis FLC6 lysed not only the B. glumae and B. plantarii strains listed in Table 1, but also the R. pseudosoranacearum strain, demonstrating that the strain has a host range that includes multiple genera of plant pathogenic bacteria.

[0059] [Table 1]

[0060] Suppression of rice seedling rot by FLC6 treatment When rice seeds contaminated with B. glumae (MAFF302746) were soaked in a diluted solution of FLC6 and then sown, disease incidence was significantly suppressed compared to the control area, which was soaked in a diluted PPG medium solution to achieve equal concentrations of medium components (Figure 4). The disease intensity was 1.0 in the FLC6-treated area and 89 in the control area, resulting in a control titer of 99. Since FLC6 has hosts across genera, it is thought that FLC6 is effective in controlling a wide range of plant diseases.

Claims

1. A bacteriophage comprising a nucleic acid molecule comprising a base sequence represented by SEQ ID NO:1, or a nucleic acid molecule comprising a base sequence having 95% or more homology to the base sequence represented by SEQ ID NO:

1.

2. The bacteriophage of claim 1, which is a jumbo phage.

3. A plant disease control agent comprising the bacteriophage according to claim 1 or 2.

4. The plant disease control agent according to claim 3, wherein the plant disease is one or both of a rice seedling disease and bacterial wilt disease.

5. A process for extracting a bacteriophage population including a target bacteriophage from an organic culture medium by mixing the organic culture medium with a liquid medium. Cultivating the phage population in the presence of host cells to grow the phages of interest. Removing host cells from the culture medium A step of culturing the culture solution from which the host cells have been removed in a soft agar medium to which the host cells have been added, to form phage plaques that infect the host cells. A method for producing a bacteriophage that infects a host cell, comprising:

6. Mixing organic soil with liquid medium A step of centrifuging and / or filtering the mixture of organic soil and liquid medium obtained in the above step. A step of adding host cells to the supernatant or filtrate obtained in the above step and culturing them. A step of centrifuging and / or filtering the culture solution obtained in the above step. A step of culturing the supernatant or filtrate obtained in the above step using a soft agar medium containing host cells. A method for producing a bacteriophage that infects a host cell, comprising:

7. The method according to claim 5 or 6, wherein the host cell is at least one species selected from the group consisting of bacteria of the genus Burkholderia and bacteria of the genus Ralstonia.

8. A method according to any one of claims 5 to 7 for obtaining a bacteriophage according to claim 1 or 2.

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