JP4862154B2 - Bacterial blight of bacterial wilt disease - Google Patents

Description

The present invention relates to a bacteriophage (hereinafter also simply referred to as “phage”) exhibiting lytic activity against bacterial wilt ( Ralstonia solanacearum ), and use thereof.

  Bacterial wilt is a soil bacterium that causes bacterial wilt (withering) in tomatoes, eggplants, tobacco, and the like. It is distributed all over the world, mainly in relatively warm regions, and infects more than 200 species of 33 families including eggplants and legumes and various economically important crops, causing tremendous damage. It is very difficult to remove pathogenic bacteria from once contaminated soil. So far, intercropping, crop rotation, soil improvement, soil disinfection methods, etc. have been devised as control methods for this disease, but their occurrence has hardly been stably suppressed.

In addition, for plant bacterial diseases, a control method using a bacteriophage that lyses pathogenic bacteria has long been expected as a means to selectively lyse and control pathogenic bacteria. For bacterial wilt disease, P4282 phage, PK101 Bacteriophages that infect bacterial wilt disease such as phages have been reported (Non-patent Documents 1 and 2). In addition, recently, the present inventors have succeeded in isolating dozens of bacteriophages that are lytic against bacterial wilt and are roughly classified into three types from the host specificity, A patent application has already been filed (Patent Document 1).
However, the bacteriophages reported so far have a narrow lysis spectrum and are sufficient for effective control of bacterial wilt disease with many strains due to host range, geographical distribution, pathogenicity, and epidemiological characteristics. is not.
JP 2005-278513 A Hiroshi Tanaka, Hideaki Negishi, Hatsue Maeda: Pseudomonas solanacearum M4S and bacteriophage control of tobacco blight, Journal of the Japanese Society for Plant Pathology, Vol. 56, No. 2, pp243-246 (1990) Toyoda H, Kakutani K, Ikeda S, Goto H, Tanaka H and Ouchi S. Characterization of deoxyribonucleic acid of virulent bacteriophage and its infectivity to host bacteria, Pseudomonas solanacearum. J. Phytopathology. (1991), 131: 11-21

  An object of the present invention is to provide means for effectively controlling bacterial wilt disease using a novel bacterial wilt infectious bacteriophage.

As a result of intensive studies in view of such circumstances, the present inventor succeeded in isolating a bacteriophage exhibiting a broad lytic spectrum infectious against bacterial wilt disease from Ralstonia solanacearum. It was found that the bacterial wilt disease can be effectively controlled by using.

That is, the present invention relates to the following (1) to (6).
(1) Isolated from Ralstonia solanacearum ( Ralstonia solanacearum ), C-319, M4S, Ps29, Ps65, Ps72, Ps74, MAFF106603, MAFF106611, MAFF211270, MAFF211272 and MAFF211272 Bacteriophage that is lytic to all of the strains MAFF301556, MAFF301558, MAFF730138 and MAFF730139.
(2) A bacterial blight control agent containing the bacteriophage as an active ingredient.
(3) A soil conditioner containing the bacteriophage as an active ingredient.
(4) A method for controlling bacterial wilt disease comprising spraying the bacteriophage on plants or soil.
(5) A method for controlling bacterial wilt disease comprising adding the bacteriophage to a culture solution of hydroponics or solution cultivation.
(6) A method for improving soil, comprising adding the bacteriophage to soil.

  The bacteriophage of the present invention exhibits a broad spectrum of action compared to the known bacterial wilt bacteriophage. Therefore, by using this, it is possible to provide an effective control agent for many bacterial wilt diseases, and it is possible to provide an effective soil conditioner for improving soil contaminated with bacterial wilt disease.

The bacteriophage of the present invention is isolated from Ralstonia solanacearum .
Ralstonia solanacearum ( Ralstonia solanacearum ) is a polycidal soil-borne bacterium that causes bacterial wilt (withering disease) on over 200 kinds of crops such as tomato, eggplant and tobacco. In the present specification, it is also referred to as “green blight fungus”.

The bacteriophage of the present invention contains at least C-319 strain, M4S strain, Ps29 strain, Ps65 strain, Ps72 strain, Ps74 strain, MAFF106603 strain, MAFF106611 strain, MAFF211270 strain, MAFF211271 strain, MAFF211272 strain of Ralstonia solanacearum , MAFF301556, MAFF301558, MAFF730138, and MAFF730139 all have lytic properties and have host specificity.
Such host specificity is different from the bacteriophages belonging to Type 1, Type 2 and Type 3 described in JP-A-2005-278513 (Patent Document 1), as shown in Example 3 below. .

Here, the M4S strain is a strain of race 1 isolated from tobacco, and is the strain described in Non-Patent Document 1.
Ps29 strain is a strain of race 1 isolated from tobacco, Ozawa H, Tanaka H, IchinoseT, Shiraishi T, and Yamada T. Bacterialphage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein, important for lytic infection of Its host. Molecular Genetics and Genomics (2001) 265: 95-101.
C-319 strain, Ps65 strain, Ps72 strain and Ps74 strain are race 1 strains isolated from tobacco.
C-319 strain, M4S strain, Ps29 strain, Ps65 strain, Ps72 strain and Ps74 strain are strains sold by San-in Construction Industry (Izumo City, Shimane Prefecture).
The MAFF106603 strain is a race 1, biovar 3 strain, the MAFF106611 strain is a race 1, biovar 4 strain, the MAFF211270 strain is a race 1, biovar N2 strain, and the MAFF211271 strain is a race 3, biovar strain. Strain N2, MAFF211272 strain is race 4, biovar 4 strain, MAFF301556 strain is race 1, biovar 4 strain, MAFF301558 strain is race 3, biovar N2 strain, MAFF730138 strain is Race 1, biovar 3 strain, and MAFF730139 strain is a strain of race 1, biovar 4. MAFF106603, MAFF106611, MAFF211270, MAFF211271, MAFF211272, MAFF301556, MAFF301558, MAFF730138, MAFF730138, and MAFF730139 are microbial genetic resources utilization manuals published by the Ministry of Agriculture, Forestry and Fisheries Bioresources Research Institute (12), Mitsuko Horita and Tsuchiya Kenichi, a strain described in March 2002, can be sold from the Genebank of Agricultural Bioresources, Ministry of Agriculture, Forestry and Fisheries.

The bacteriophage having the above host specificity (lysis spectrum) is included in the bacteriophage of the present invention. Among them, those isolated from Ralstonia solanacearum MAFF211272 strain are preferable. The MAFF211272 strain is a strain of race 4 and biovar 4 as described above, and can be sold from the Genebank of Agricultural Bioresources, Ministry of Agriculture, Forestry and Fisheries.
Of these, the bacteriophage named “RSA1” is particularly preferred.
RSA1 is stored in a state that can be sold by the applicant. The RSA1 applied for a deposit (NITE AP-210 (February 22, 2006)) at the National Institute of Technology and Evaluation (NITE), but was rejected.

Hereinafter, other characteristics of bacteriophage RSA1 will be described.
1. Form As shown in FIG. 1, bacteriophage RSA1 is composed of an icosahedral head and a tail on a line. This is because the P4282 phage described in Non-Patent Document 1 has a head diameter of about 70 nm and a tail of about 20 nm, whereas RSA1 has a head diameter of about 40 nm and a tail of about 20 nm. It differs in that it is about 100 nm.

2. Genome analysis The genome of bacteriophage RSA1 consists of double-stranded DNA, and the pattern of the fragments by the restriction enzyme ( Sma I) treatment is as shown in FIG. (Example 5).
The genome size of RSA1 determined by electrophoresis is about 39 Kbp (FIG. 2), which is different from PK101 phage (about 35 Kbp) of Non-Patent Document 2.
In addition, when the base sequence of RSA1 was determined, the size was 38,820 bp, and the region of the Ralstonia solanacearum GMI1000 strain, which has been determined for the entire genome, shows a very high homology with the RSA1 sequence. (FIG. 3), indicating that RSA1 may be lysogenized and retained in the genome of Ralstonia solanacearum .
From the above, it can be said that bacteriophage RSA1 is a novel bacterial wilt infectious bacteriophage not described in the literature.

The bacteriophage of the present invention can be isolated from bacterial wilt by using a plaque assay / separation method. That is, for example, Ralstonia solanacearum ( Ralstonia solanacearum ) MAFF211272 strain is swirled in CPG (Example 1) medium, and this culture is mixed with top agar and spread on a CPG agar medium. Incubate at 25-30 ° C for 12-48 hours, pick up the central part of the detected single plaque with a sterile toothpick, etc., and add to a culture solution such as Ralstonia solanacearum M4S strain to amplify the bacteriophage Let me. After amplification, the bacteriophage RSA1 can be separated by sterilizing the centrifuged supernatant with a 0.2 μm pore filter or the like.

The bacteriophage of the present invention can be grown in the same manner as a general bacteriophage. For example, a large amount of phage solution can be obtained by culturing and fully growing Ralstonia solanacearum , inoculating the bacteriophage of the present invention, and continuing the culture. Cultivation of Ralstonia solanacearum ( Ralstonia solanacearum ) can be performed according to a conventional method, for example, using a CPG liquid medium or the like, and culturing at a temperature of 27 to 37 ° C. with swirling or shaking. The time to inoculate the bacteriophage is not particularly limited as long as Ralstonia solanacearum is sufficiently proliferating, but the time when the cell concentration has increased to about 1 × 10 ⁴ to 1 × 10 ⁸ cfu / ml It is desirable to inoculate. By culturing for 6 to 12 hours after phage inoculation, almost all bacteria are lysed, and a phage solution can be obtained.

  As described above, the bacteriophage of the present invention has a broad action spectrum (Example 3) as compared with known bacterial blight infectious bacteriophages, and is excellent when applied to fields, hydroponics and the like. (Example 8). Therefore, this bacteriophage can be used for controlling bacterial wilt disease, improving soil contaminated with bacterial wilt disease, and controlling the bacteria in hydroponics and solution cultures. The term “control” used herein includes both “control” of infected bacterial wilt and “prevention” of infection.

  When the bacteriophage of the present invention is used as a bacterial wilt control agent or a soil conditioner, a phage solution obtained by the growth method described above, or a phage solution separated from the culture residue by centrifugation or the like may be used. Although it is good, even if it uses as a solution which mix | blended the spreading agent, it can adhere to a plant body and is preferable. In addition, a solid carrier or a liquid carrier generally used in microbial preparations may be blended to prepare a preparation form such as a wettable powder, powder, capsule or the like.

In the control agent or soil conditioner containing the bacteriophage, the phage concentration in the solution is preferably 1 × 10 ⁵ to 1 × 10 ¹¹ pfu / ml, and 1 × 10 ⁸ to 1 × 10 ¹⁰ pfu. More preferably / ml.

Examples of plants to be controlled include, but are not limited to, potato, eggplant, tomato, pepper, pepper, tobacco, perilla, radish, strawberry, banana, margaret, chrysanthemum, sunflower and the like. . The disease to be controlled is not limited as long as it is a disease caused by Ralstonia solanacearum , and examples thereof include bacterial wilt of solanaceous plants and tobacco wilt.

When the control agent of the present invention is sprayed on the field, the spraying amount is not particularly limited as long as it is within the range where bacterial wilt can be controlled, but usually 1 × 10 ⁸ to 1 × 10 ¹² pfu phage per square meter of the field It is preferable to spray. When used for control in hydroponics or solution culture, it is preferable to add phages to the culture solution so as to be 1 × 10 ³ to 1 × 10 ¹⁰ pfu / ml.

Further, when the soil improving agent of the present invention is added to the soil, the amount added is not particularly limited, it is preferable usually to add the soil 1kg per ^{1 × 10 10 ~1 × 10 12} pfu of the phage.

  Hereinafter, the present invention will be described in more detail with reference to examples. Unless otherwise stated, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001) ; FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. The method, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

Example 1 Culture of bacterial wilt fungus CPG liquid medium was prepared by adding 10.0 g of polypeptone, 1.0 g of casamino acid, and 5.0 g of glucose to 1,000 ml of distilled water. Using this CPG liquid medium, each bacterial strain of bacterial wilt was cultured at 28 ° C. by rotating culture (200 rpm) or shaking culture (300 rpm).

Example 2 Separation of Bacteriophage RSA1 Bacteriophage RSA1 was isolated from the bacterial wilt fungus MAFF211272 by a plaque assay / separation method.
Ralstonia solanacearum ( Ralstonia solanacearum ) MAFF211272 strain was swirled in CPG medium, 250 μL of this culture was mixed with 4 ml of top agar, and then on CPG agar medium (medium with 17.0 g of agar added to the above CPG liquid medium) Spread out. Plaques were detected after culturing at 28 ° C. for 12 to 48 hours. The central part of the single plaque was picked with a sterile toothpick and added to a culture solution (OD600 = 0.1 to 0.5) of bacterial wilt (M4S strain) to amplify the bacteriophage. After amplification, the centrifuged supernatant was sterilized by filtration with a 0.2 μm pore filter to separate bacteriophage RSA1.
This phage RSA1 may have been lysogenized in the bacterial wilt fungus MAFF211272.
RSA1 applied for deposit at the Patent Microorganisms Depositary Center for Product Evaluation and Technology on February 15, 2006 (Receipt Number NITE AP-210 (February 22, 2006)). On March 28, 2018, the acceptance was refused.

Example 3 Host specificity of bacteriophage RSA1 Host specificity of bacteriophage RSA1 against bacterial strains of bacterial wilt was examined by a plaque assay method. The bacteriophage RSA1 isolated in Example 2 was mixed with the culture solution (OD600 = 0.1 to 0.5) of each bacterial wilt of bacteria obtained in Example 1, and incubated at 28 ° C. for 1 hour, and then 4 ml of top agar And spread on a CPG agar medium, and plaque assay was performed. Ralstonia solanacearum ( Ralstonia solanacearum ) C319 strain, M4S strain, Ps29 strain, Ps65 strain, Ps72 strain, Ps74 strain, MAFF106603 strain, with different pathogenicity, epidemiological characteristics, physiological and biochemical properties MAFF106611 strain, MAFF211270 strain, MAFF211271 strain, MAFF211272 strain, MAFF301556 strain, MAFF301558 strain, MAFF730138 strain, MAFF730139 strain were used.
In order to compare and examine the homology with three types of bacteriophages (Patent Document 1) already isolated from bacterial wilt disease by the present inventor, L-type (type 1 phage), M-type (type 3) The same experiment was performed on bacteriophages of phage) and S-type (type 2 phage). The results are shown in Table 1.

  As shown in Table 1, bacteriophage RSA1 had all the strains used in the assay as hosts, and the host specificity was different from the three types of already isolated bacteriophages (Patent Document 1).

Example 4 Morphological Observation of Bacteriophage RSA1 The morphology of bacteriophage RSA1 was observed using a transmission electron microscope. 10 ml of bacteriophage RSA1 filter sterilized solution shown in Example 2 was centrifuged at 40,000 g for 1 hour to obtain bacteriophage RSA1 as a precipitate. This precipitate was suspended in 100 μl of PBS containing 0.01 mol / l MgSO ₄ . The bacteriophage suspension was dropped onto a grid covered with a formbar film, negatively stained with phosphotungstic acid, and then observed with a transmission electron microscope. The result is shown in FIG.

As shown in FIG. 1, bacteriophage RSA1 was composed of an icosahedral head and a linear tail. The tail end was thick. This form was different from that reported in Non-Patent Document 1. There are no reports of similar phages that infect Ralstonia solanacearum in the literature.

Example 5 Analysis of Genome of Bacteriophage RSA1 Nucleic acid was isolated from bacteriophage RSA1 and analyzed. The genome of RSA1 consists of double-stranded DNA, and the genome size of phage RSA1 was determined by electrophoresis, and it was about 39 kbp as shown in FIG. Analysis of the genome of RSA1 from the electrophoresis pattern of untreated DNA purified from RSA1 (Figure 2A) and DNA after digestion with Sma I (Figure 2B) (In Figure 2A, a low-concentration agarose gel was used. The DNA of 20 Kbp or more was separated by electrophoresis), and the fragment pattern was different from the phages of Patent Document 1 and Non-Patent Document 2 (FIG. 2).

Example 6 Determination of the genomic sequence of bacteriophage RSA1
When the base sequence of RSA1 was determined, the size was 38,820 bp. As shown in FIG. 3, it was found that a region having very high homology with the phage RSA1 sequence was present in the genome of Ralstonia solanacearum GMI1000 strain (the entire genome was determined).
The presence of a region highly homologous to the phage RSA1 genome in the genome of Ralstonia solanacearum GMI1000 strain indicates that RSA1 can be lysed and retained in the Ralstonia solanacearum genome. Showing sex.

Example 7 Mass production of bacteriophage RSA1
A single colony of Ralstonia solanacearum M4S strain cultured on CPG agar medium was inoculated into 50 ml of CPG liquid medium and swirled at 28 ° C. for 24-48 hours. 10 ml of this culture solution was added to 2,000 ml of CPG liquid medium, and swirling culture (120 rpm) was performed at 28 ° C. for 4 to 6 hours until OD600 = 0.1 (about 5 × 10 ⁷ cfu / ml). To this culture solution, 10 to 100 ml of a phage solution diluted in advance so that moi (abbreviation of The multiplicity of infection, number of phages per cell) = 0.0001 to 0.1 was added. For the dilution of the phage, a solution in which MgSO ₄ was added to the CPG liquid medium shown in Example 1 to a final concentration of 0.01 mol / l was used. After standing at 28 ° C. for 1 hour, swirling culture was performed at 28 ° C. for 10 hours or more (120 rpm), and a large amount of bacteriophage solution was prepared by lysis. The lysate contained 5 × 10 ^{7 to} 5 × 10 ⁹ pfu / ml of phage RSA1.

Example 8 Bacteriophage RSA1 inhibits growth of bacterial wilt
A single colony of Ralstonia solanacearum ( Ralstonia solanacearum ) M4S strain cultured on a CPG agar medium was inoculated into 50 ml of a CPG liquid medium, and swirled at 28 ° C. for 24 hours. 0.5 ml of this culture solution was added to 100 ml of CPG liquid medium, and swirl culture (200 rpm) was performed at 28 ° C. for 4 to 6 hours until OD600 = 0.1 (about 5 × 10 ⁷ cfu / ml). To this culture solution, 20 μl of a phage RSA1 solution diluted in advance to a final concentration of 0.01 mol / l MgSO _{4 in} CPG liquid medium was added, and moi (abbreviation of The multiplicity of infection, per cell) The number of phages against each other was 0.01, 0.001, 0.0001. Control samples were not infected with phage. After standing at 28 ° C. for 30 minutes, culturing was continued at 28 ° C. for 16 hours (200 rpm), OD600 was measured every 2 hours, and growth of bacterial wilt disease was recorded. The results are shown in FIG.

  Addition of phage RSA1 significantly inhibited the growth of bacterial wilt. The degree of growth inhibition depended on m.o.i. at the time of phage infection, and the larger m.o.i., the stronger the growth inhibition effect.

[Mode 1] Control of bacterial wilt disease in a field When spraying on a field, 1 × 10 ⁸ to 1 × 10 ¹² pfu of phage per 1 square meter of the field is sprayed. When mixed with soil, 1 × 10 ¹⁰ to 1 × 10 ¹² pfu of phage per 1 kg of soil is mixed with the soil and used.

[Mode 2] Control of bacterial wilt fungus in hydroponics and solution culture Using phage RSA1, bacterial wilt fungus is controlled in hydroponics or solution culture. Phage RSA1 is added to a culture solution used in hydroponics or solution cultivation so that the concentration becomes 1 × 10 ³ to 1 × 10 ¹⁰ pfu / ml.

It is an electron microscope observation photograph by the negative staining method of RSA1. The scale bar indicates 100 nm. It is a photograph which shows the electrophoresis pattern of DNA refine | purified from RSA1 (Aphi). Fig. 2A: Untreated DNA purified from RSA1, Fig. 2B: DNA after Sma I digestion. λ, λ / Sty I, λ / Sty I + λ / Bgl II, each lane indicated by λ / Hin dIII + φX174 / Hae III size marker. The figure which shows the homology of the genome sequence of bacteriophage RSA1 and the genome sequence of Ralstonia solanaceram GMI1000 strain. FIG. 3A: Physical map obtained from the base sequence of the genome of phage RSA1 (full length 38.8 kbp). Terminals ◯ and □ represent both ends of the phage genomic DNA. FIG. 3B: There is a homology between the genome sequence of phage RSA1 and the genome of Ralstonia solanacelam GMI1000, and the corresponding part is shown. FIG. 3B top: RSA1 genome shows the structure of the genome assuming that both ends are linked to form a circular structure in bacterial wilt. Fig. 3B bottom: Ralstonia Solanacerum GMI1000 strain physical map. Upper number: The distance from the terminal (◯) of phage RSA1 is expressed in Kbp. Lower number: The distance from the end (▲) of the genome of GMI1000 strain in Kbp. It is a figure which shows the growth inhibitory effect of bacterial wilt disease by bacteriophage RSA1.

Claims (6)

Ralstonia solanacearum (Ralstonia solanacearum) isolated from MAFF211272 strain, Ba click bacteriophage, designated RSA1. Control agent of blue blight containing bacteriophage according to claim 1 Symbol placement as an active ingredient. Soil improver containing the bacteriophage of claim 1 Symbol placement as an active ingredient. Blue blight control method, wherein a bacteriophage according to claim 1 Symbol placement sprayed to plants or soil. Blue blight control method, characterized in that the addition of bacteriophage according to claim 1 Symbol placement in culture water culture or solution cultivation. Improved method of soil, wherein a bacteriophage according to claim 1 Symbol placement added to the soil.