WO2015194540A1

WO2015194540A1 - Bacteriophage, agent for preventing xanthomonas campestris disease, and method for preventing xanthomonas campestris disease

Info

Publication number: WO2015194540A1
Application number: PCT/JP2015/067282
Authority: WO
Inventors: 山田　隆; 藤江　誠; 川崎　健
Original assignee: 国立大学法人広島大学
Priority date: 2014-06-20
Filing date: 2015-06-16
Publication date: 2015-12-23
Also published as: JPWO2015194540A1

Abstract

　A bacteriophage, having phage particles of linear structure, infects Xanthomonas campestris pv. citri. This bacteriophage may have a genome size of 7,000-8,000 b. This bacteriophage may have a genome configuration including a DNA replication module, a structural protein module, an assembly and secretion module, and a regulatory region.

Description

Bacteriophage, citrus canker prevention agent and citrus canker prevention method

The present invention relates to a bacteriophage, a citrus canker prevention agent, and a method for preventing citrus canker.

Copper chemicals are the main agricultural chemicals used to combat citrus canker disease that affects citrus. For example, Cited Document 1 discloses a plant treatment composition containing a copper agent and an amine compound. As another agrochemical, a plant disease control agent containing signamycin as an active ingredient is disclosed in Citation 2. Furthermore, cited reference 3 discloses a plant disease control agent using a non-pathogenic Xanthomonas bacterium as a biopesticide that can be used for measures against citrus canker.

Special table 2013-536237 gazette JP 2010-222292 A JP 2013-215189 A

Since the copper preparation contained in the plant treatment composition disclosed in Patent Document 1 is a deleterious substance, there is a concern about the effect of residual agricultural chemicals on the health when used in food crops. In addition, some of the used copper agent is washed away into the river, groundwater or sea by rain, so there is a risk of environmental pollution. Furthermore, an increase in citrus scab disease resistant to copper agents is a problem.

As for the control agent containing signamycin disclosed in Patent Document 2 as an active ingredient, an effective application amount is expected to increase due to the emergence of resistant citrus canker as long as it is a chemical pesticide.

The control agent using a non-pathogenic Xanthomonas genus bacterium disclosed in Patent Document 3 is not specific to antagonizing bacteria and the like, and may affect other useful microorganisms.

The present invention has been made in view of the above circumstances, and provides a bacteriophage, a citrus canker prevention agent, and a method for preventing citrus canker that are highly specific for citrus canker and can prevent citrus canker more safely. The purpose is to do.

The bacteriophage according to the first aspect of the present invention is:
The structure of the phage particle is linear and infects citrus canker.

In this case, the genome size is 7000-8000b.
It is good as well.

In addition, the genome structure includes a DNA replication module, a structural protein module, an association / secretion module, and a control region.
It is good as well.

Further, the genome base sequence consists of the base sequence shown in SEQ ID NO: 1.
It is good as well.

In addition, the citrus canker is Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. citri, or Xanthomonas citri,
It is good as well.

The citrus canker prevention agent according to the second aspect of the present invention,
The bacteriophage according to the first aspect of the present invention is included.

The citrus canker prevention agent according to the third aspect of the present invention,
The citrus scab pathogen infected with the bacteriophage according to the first aspect of the present invention is included.

In this case, the citrus canker is Xanthomonas campestris pv. Citri MAFF301080 strain infected with the bacteriophage (name of depositary organization: National Institute of Technology and Evaluation, Patent Microorganism Depositary, Address of depositary organization: 2 Kazusa Kamashishi, Kisarazu City, Chiba Prefecture, Japan 292-0818 -5-8 Room 122, Deposit Date: June 16, 2014, Deposit Number: NITE BP-01875)
It is good as well.

The method for preventing citrus canker disease according to the fourth aspect of the present invention includes:
Administering the citrus scab prevention agent according to the second aspect of the present invention or the citrus scab prevention agent according to the third aspect of the present invention to a plant or a plant growth medium.

In this case, the plant is a citrus.
It is good as well.

According to the present invention, the specificity to citrus canker disease is high, and citrus canker disease can be prevented more safely.

It is a figure which shows an example of the shape of the bacteriophage which concerns on this invention. It is a figure which shows the time-dependent change of the proliferation of the bacteriophage which concerns on this invention. It is a figure which shows the structure of the genome of the bacteriophage which concerns on this invention. It is a figure which shows the Swimming motility of the strain infected with the bacteriophage which concerns on this invention. (A) is a figure which shows the colony of an uninfected host strain. (B) is a diagram showing colonies of strains infected with bacteriophages. (C) is a figure which shows the time-dependent change of the motility distance of the uninfected host strain and the strain infected with the bacteriophage. It is a figure which shows the Swarming motility of the strain infected with the bacteriophage which concerns on this invention. (A) is a figure which shows the colony of an uninfected host strain. (B) is a diagram showing colonies of strains infected with bacteriophages. (C) is a figure which shows the time-dependent change of the motility distance of the uninfected host strain and the strain infected with the bacteriophage. It is a figure which shows Twitching motility of the strain infected with the bacteriophage which concerns on this invention. (A) is a figure which shows the colony of an uninfected host strain. (B) is a diagram showing colonies of strains infected with bacteriophages. It is a figure which shows the state which precipitated the microbial cell by centrifugation after liquid culture | cultivating the bacterial strain which infected the bacteriophage which concerns on this invention, and an uninfected host strain, respectively. It is a figure which shows the time-dependent change of the leaf which inoculated the strain infected with the bacteriophage which concerns on this invention by the needlestick method. (A) is a figure which shows the time-dependent change of the leaf which inoculated the bacteriophage uninfected host strain. (B) is a figure which shows the time-dependent change of the leaf inoculated with the strain infected with the bacteriophage. It is a figure which shows the magnitude | size of the lesion in the leaf which inoculated the strain infected with the bacteriophage which concerns on this invention with the needle stick method. It is a figure which shows the time-dependent change of the leaf which inoculated by the osmosis | permeation method the strain infected with the bacteriophage which concerns on this invention. It is a figure which shows the time-dependent change of the leaf which sprayed the strain infected with the bacteriophage which concerns on this invention. (A) is a figure which shows the time-dependent change of the leaf which sprayed the bacteriophage uninfected host strain. (B) is a figure which shows the time-dependent change of the leaf which sprayed the strain infected with the bacteriophage. It is a figure which shows the citrus canker prevention effect by the strain infected with the bacteriophage which concerns on this invention. (A) is a figure which shows the time-dependent change of the leaf which inoculated the citrus canker disease bacteria without spraying the bacteriophage infected strain. (B) is a figure which shows the time-dependent change of the leaf which inoculated the citrus canker disease fungus after spraying the strain which the bacteriophage infected.

Embodiments according to the present invention will be described with reference to the accompanying drawings. In addition, this invention is not limited by the following embodiment and drawing.

(Embodiment 1)
The citrus scab prevention agent according to the present embodiment includes a citrus scab fungus infected with bacteriophage. The bacteriophage has a linear phage particle structure and infects citrus canker. The bacteriophage can be said to be an Inovirus or Ff type phage because the structure of the phage particle is linear. That the structure of the phage particle is linear means that the structure of the phage particle is as shown in FIG. The structure of the phage particle can be observed with an electron microscope by negatively staining the phage particle with sodium phosphotungstate.

The size of the phage particle depends on the genome size. When the genome size is 7000 to 8000b, the length of the phage particle is approximately 8000 to 1500 nm. In the case of the phage particle shown in FIG. 1, the phage particle has a length of 1000 nm and a diameter of 7 nm.

The genome size of the bacteriophage shown in FIG. 1 is 7325b, but the genome size of the bacteriophage is not limited to this. The genome size of the bacteriophage may be 7000-8000b, or 7000-7600b, preferably 7100-7500b, more preferably 7200-7400b.

The base sequence of the bacteriophage genome shown in FIG. 1 consists of the base sequence shown in SEQ ID NO: 1. The genome structure of the bacteriophage includes a DNA replication module, a structural protein module, and an association / secretion module as common modules with E. coli M13, which is a representative of Inovirus (Ff type phage). Furthermore, the genomic organization of the bacteriophage includes a regulatory region downstream of the association / secretion module.

The base sequence of the bacteriophage genome can be determined by a known method such as a shotgun sequencing method after DNA is isolated from the bacteriophage by phenol extraction or the like. By analyzing the determined base sequence of the genome using a sequence analysis program and a sequence database, the functions of the ORF and the gene can be estimated.

The bacteriophage is not limited to the base sequence shown in SEQ ID NO: 1, but is a bacteriophage that has a linear phage particle structure and infects citrus canker. And 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% sequence homology.

The citrus canker that is infected by the bacteriophage is a pathogen that causes diseases on citrus fruits, leaves, branches, and the like. Citrus canker can be obtained by, for example, Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. citri, or Xanthomonas citri. Citrus canker disease is, for example, the MAFF strain available from the National Institute for Agrobiological Sciences. More specifically, the citrus canker that is infected with bacteriophage is MAFF301080, MAFF302102, MAFF673011, MAFF673030, MAFF6733013, MAFF6730301, and the like.

The bacteriophage can be obtained, for example, by washing and centrifuging a sample containing soil and filtering the supernatant with a membrane filter. Furthermore, the target bacteriophage can be isolated by using the above citrus scab pathogen suitable as a host. For isolation of bacteriophage and measurement of titer, a method of forming plaques by overlaying an agar medium with a soft agar medium (0.75% agar) obtained by adding a test bacteria and a phage sample mixed solution to the agar medium is preferable.

As the method for infecting bacteriophage with citrus canker, any method known in the art can be used. As an example, bacteriophage can be infected with citrus canker by adding bacteriophage to a culture solution containing citrus canker that is cultured in an NB (Nutrient Broth) medium.

The citrus canker that is infected with bacteriophage grows while producing and secreting the bacteriophage without lysis. In order to confirm that the citrus canker that is infected with the bacteriophage is growing without lysis, the absorbance of light having a wavelength of 600 nm, which is an indicator of the concentration of the bacterium, may be measured.

As one of the important virulence factors of citrus canker disease, exopolysaccharides (also referred to as “EPS”) such as xanthan are known. As shown in Example 5 below, infection with bacteriophages greatly reduces the production of extracellular polysaccharides, particularly xanthan, from citrus canker. For this reason, as shown in Example 6 below, bacteriophage infects pathogenic citrus canker disease bacteria, thereby significantly reducing the pathogenicity of the citrus itchworm fungus and eliminating the ability to form itch.

In addition, the citrus canker that is infected with the bacteriophage has a decrease in at least one of the swimming motility, the swarming motility, and the switching motility as compared with an uninfected citrus canker (see Example 4 below). In addition to the significant reduction in exopolysaccharide production described above, this reduction in motility contributes to a marked reduction in the pathogenicity of bacteriophage-infected citrus canker and the loss of ability to form itch.

As the citrus scab disease fungus contained in the citrus scab disease prevention agent according to the present embodiment, for example, Xanthomonas campestris pv. Citri MAFF301080 strain infected with the bacteriophage (name of depositary organization: National Institute of Technology and Evaluation, Patent Microorganism Depository Center, name of depositary organization: 292-2818, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan -5-8 Room 122, Deposit Date: June 16, 2014, Deposit Number: NITE BP-01875).

The citrus canker prevention agent according to the present embodiment may be administered to a plant or a plant growth medium such as soil, a mat, a solid medium or the like, a nutrient solution in hydroponics, and water in hydroponics. By carrying out like this, the bacteriophage secreted by the citrus canker disease contained in the citrus canker disease preventive agent can be infected with a potential pathogenic citrus canker disease. As a result, citrus canker can be prevented. Here, “prevention” refers to prevention of plant infection with citrus scab, prevention of plant disease with citrus scab, prevention of spread of plant illness with citrus scab, and control of pathogenic citrus scab. Including. The plant growth medium may be any medium as long as the plant grows.

The plant to which the citrus canker disease preventive agent is administered may be of any kind as long as it can be affected by the disease of citrus canker disease, and in particular, citrus. By administering the citrus scab disease preventive agent to the plant growth medium by spraying or the like, it is possible to prevent the plant growing in the plant growth medium from being infected with citrus scab disease fungus or the disease caused by citrus scab disease fungus. In addition, by administering the citrus canker disease preventive agent to a plant by spraying or the like, if the plant is not infected with pathogenic citrus canker disease, the plant may be infected with citrus canker disease or citrus canker disease. Diseases can be prevented from reaching the plant. If the plant is infected with a pathogenic citrus canker, it can be prevented from spreading by administering the citrus canker preventive.

The citrus canker disease preventive agent according to the present embodiment includes citrus canker disease fungus infected with bacteriophage at any concentration as long as a desired effect is obtained. For example, citrus scabs infected with bacteriophages in suspension in sterile water can be 10 ³ to 10 ¹⁴ colony forming units (cfu) / mL, 10 ⁴ to 10 ¹² cfu / mL or 10 ³ to 10 ⁸ cfu. / ML. When administered to plants at this concentration, the citrus scab prevention agent may be administered in an amount of 1 μL to 1000 mL, 10 μL to 100 mL, 100 μL to 10 mL, or 1 to 5 mL per plant individual, and in any amount higher than this. May be. When administering to a plant, the said citrus scab prevention agent may be put into a syringe and inoculated by pressure, or may be inoculated via an injection needle. When a suspension containing the citrus canker prevention agent is administered to a plant growth medium, for example, 1 μL to 1000 mL, 10 μL to 100 mL, 100 μL to 10 mL, or 1 to 5 mL is sprayed per 1 m ² of the surface area of the plant growth medium. May be administered in any amount greater than this.

The citrus canker prevention agent according to the present embodiment may be administered once to a plant or a plant growth medium, or may be administered multiple times to a plant or a plant growth medium at an arbitrary time interval. As shown in Example 6, the citrus scab prevention agent maintained a significant decrease in pathogenicity and loss of itch formation ability for 4 weeks after administration to plants. For this reason, the citrus canker prevention agent according to the present embodiment is applied to plants or the like at intervals of once or several times a week, once every two weeks, once every three weeks, once every four weeks, etc. What is necessary is just to administer. The administration interval can be appropriately determined according to the plant to be administered or the plant growth medium.

The citrus canker prevention agent according to the present embodiment may include other pharmaceutically or botanically acceptable substances, compositions, and the like in addition to the citrus canker disease fungus infected with the active ingredient bacteriophage. Good.

As described above in detail, the citrus scab disease preventive agent according to the present embodiment can remarkably reduce the pathogenicity of citrus scab disease fungi, and can eliminate the itch formation ability. For this reason, the plant can be prevented from being infected with citrus canker disease, or the onset or spread of citrus canker disease in the plant can be prevented. Moreover, the said citrus canker disease preventive agent can be used also for control of a citrus canker disease fungus.

In addition, since the bacteriophage secreted by the citrus canker disease contained in the citrus canker disease preventive agent according to the present embodiment has a property of specifically infecting the citrus canker disease, it has high specificity and other useful microorganisms. Does not affect. Thereby, the said citrus canker prevention agent can make the influence on an environment small as much as possible. Therefore, the citrus canker prevention agent is safer than chemical pesticides and can avoid problems such as environmental pollution and residual pesticides.

In addition, since the citrus scab prevention agent according to the present embodiment has the bacteriophage in the citrus scab, it is possible to reduce the influence of environmental factors such as ultraviolet rays, drying and temperature changes on the bacteriophage. That is, bacteriophage stability and persistence can be increased. For this reason, the citrus canker prevention agent which concerns on this Embodiment can prevent a citrus canker disease over a long period of time. In addition, the citrus scab fungus contained in the citrus scab disease preventive agent produces a bacteriophage and secretes the produced bacteriophage while proliferating, and thus further infects the potential pathogenic citrus scab fungus be able to. Thereby, it is possible to prevent citrus canker disease with a small amount of administration, and it can also be used as a non-pathogenic antagonist. Therefore, the citrus canker prevention agent according to the present embodiment improves the convenience of plant producers and contributes to the provision of safe agricultural products at low cost.

The above bacteriophage can also be used as a preventive agent for citrus canker disease. For example, a citrus scab prevention agent comprising a bacteriophage suspended in an appropriate solvent as an active ingredient is administered to a plant or a plant growth medium, so that the bacteriophage is transmitted to a potential pathogenic citrus scab. Can infect and prevent citrus canker disease.

The dose of the citrus scab prevention agent containing the bacteriophage as an active ingredient can be determined as appropriate according to the type of plant to be administered, the volume of the plant growth medium, and the like. The dose of bacteriophage contained in a citrus canker prevention agent is measured, for example, in infectious units, where the infectious units are defined as plaque forming units (pfu), which is the ability to form clear areas or plaques on a bacterial culture plate. . For example, a suitable dose is 10 ² to 10 ¹⁰ pfu per plant individual, preferably 10 ⁴ to 10 ⁸ pfu. The citrus canker prevention agent contains the above-mentioned dose of bacteriophage in a suitable carrier or diluent, and is, for example, 100 μL to 100 mL or 1 to 10 mL depending on the type of plant to be administered or the volume of the plant growth medium. .

According to the above-mentioned citrus canker disease preventive agent containing bacteriophage as an active ingredient, since bacteriophage is infected with citrus canker disease and self-growth, citrus canker disease can be prevented with a small amount of administration.

In addition, as another embodiment of the bacteriophage, there is a production inhibitor of extracellular polysaccharides of citrus scab. The extracellular polysaccharide production inhibitor contains the bacteriophage as an active ingredient. By exposing pathogenic citrus canker disease bacteria to exopolysaccharide production inhibitors by any method, the above bacteriophage can infect citrus canker disease bacteria and suppress the production of exocytosis polysaccharides by citrus canker disease bacteria. it can. By carrying out like this, the pathogenicity of a citrus scab can be remarkably reduced and the ability to form a scab can be eliminated.

Furthermore, as another embodiment, there is a method for suppressing the production of exopolysaccharides of citrus canker. The method for inhibiting the production of exopolysaccharide includes a step of infecting the bacteriophage with a citrus scab.

(Embodiment 2)
Next, the second embodiment will be described. The method for preventing citrus canker disease according to the present embodiment includes the step of administering the agent for preventing citrus canker according to Embodiment 1 to a plant or a plant growth medium. The plant may be of any kind as long as it can be affected by the disease of citrus canker, but is preferably citrus. More preferably, the plant is a high-grade citrus such as lemon or navel orange.

The method for administering the citrus canker disease preventive agent is arbitrary as long as it can expose the plant or plant growth medium to the citrus canker disease preventive agent. The method for administering the citrus canker prevention agent includes, for example, spraying and injecting the citrus canker disease preventing agent, or allowing the citrus canker disease preventing agent to penetrate into a plant or a plant growth medium.

The dose of the citrus scab prevention agent is arbitrary, but the citrus scab prevention agent containing citrus scab disease bacteria infected with bacteriophages at an arbitrary concentration is 1 μL to 1000 mL, 10 μL to 100 mL, 100 μL to 10 mL, 1-5 mL may be administered. Alternatively, the citrus canker prevention agent may be administered by spraying 1 μL to 1000 mL, 10 μL to 100 mL, 100 μL to 10 mL, or 1 to 5 mL per 1 m ² of the surface area of the plant growth medium.

As described above in detail, the method for preventing citrus canker disease according to the present embodiment can efficiently cause a bacteriophage that significantly reduces the pathogenicity of citrus canker disease to a plant or a plant growth medium.

The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Isolation and purification of bacteriophage
The MAFF strain, which is a citrus canker disease used in the test, was sold by the National Institute for Agrobiological Sciences. The MAFF strain was cultured at 28 ° C. using a nutrient agar medium (NA medium; Difco ™, manufactured by BBL Becton Dickinson and Company, Cockeys Hill, MD, USA). When liquid culture was required, shaking culture was performed at 220 ° C. for 24 hours in NB medium (BBL Becton Dickinson & Company) (220 rpm). The MAFF strain was stored at −80 ° C. in 0.8% NB medium containing 30% (v / v) glycerol.

A linear bacteriophage (hereinafter also simply referred to as “phage”) was detected from a soil sample collected from a Japanese farmland as follows. Approximately 10 g of soil sample was placed in a sterile 50 mL conical centrifuge tube, which was filled with tap water and inverted every 20 minutes. Next, the tube was centrifuged at 1500 × g for 20 minutes, and the supernatant was filtered using a membrane filter (Millipore, Bedford, Mass., USA) having a membrane pore diameter of 0.45 μm.

Next, 100 μL of the soil filtrate is mixed with soft agar medium (0.75% agar) using MAFF strain as a host, and overlaid on an NB plate containing 1.5% (w / v) agar to form a plaque. I let you. Phages were purified from those grown on a single plaque. A bacterial culture grown overnight in 1 mL of NB medium was diluted 100-fold with 100 mL of fresh NB medium in a 500 mL flask. In order to obtain a sufficient amount of phage particles, bacterial culture was performed with a total of 2 L of culture solution. When the OD _{600 nm} reached 0.2, phages were added so that the MOI (multiplicity of infection) was 0.001 to 1.0. After further culturing for 12 to 24 hours, the cells were removed by centrifugation (8000 × g, 4 ° C. for 15 minutes using an R12A2 rotor) with a centrifuge (Hitachi Himac CR21E).

The supernatant was filtered through a membrane filter having a membrane pore diameter of 0.45 μm, and phage particles were precipitated in the presence of 0.5 M NaCl and 5% (v / v) polyethylene glycol 6000 (manufactured by Kanto Chemical Co., Inc.). . The pellet was recovered by centrifugation (15000 × g, 4 ° C. for 30 minutes using RPR20-2 rotor) with a centrifuge (Hitachi Himac CR21E), and the pellet was collected in SM buffer (50 mM Tris / HCl (pH 7. 5), 100 mM NaCl, 10 mM MgSO ₄ and 0.01% gelatin (w / v)). When the phage was stored, it was left in a light-shielding environment at 4 ° C.

The titer of the phage was determined by serial dilution and plaque formation test. 5 μL of the purified phage (10 ¹³ pfu / mL) was dropped on a mesh covered with a carbon support membrane, negatively stained with 5 μL of 2% sodium phosphotungstate, and observed with an electron microscope (Hitachi H600A).

(result)
FIG. 1 shows an electron microscopic image of the obtained phage (hereinafter referred to as “XacF1 phage”) particles. The particles had a linear structure with a length of 1000 nm and a diameter of 7 nm.

Table 1 shows XacF1 phage hosts. XacF1 phage was confirmed to infect at least 6 strains of citrus canker that infect different citrus varieties.

(Example 2: proliferation test)
In order to examine the growth of the MAFF strain infected with the XacF1 phage, the MAFF strain infected with the XacF1 phage (hereinafter also simply referred to as “XacF1 infected strain”) and the uninfected MAFF strain were cultured overnight in 5 mL of NB medium. Subsequently, 0.5 mL of MAFF strain suspension (10 ⁸ cfu / mL) was placed in a 100 mL flask containing 30 mL of NB medium. The flask was incubated at 28 ° C. with stirring at 200 rpm, and OD _{600 nm} was measured every 3 hours for 48 hours using a spectrophotometer.

(result)
FIG. 2 shows the time course of OD _{600 nm} . The MAFF strain infected with XacF1 phage (hereinafter, also simply referred to as “XacF1 infected strain”) has been shown to grow while producing XacF1 phage without lysis because OD _{600 nm} is increased. The XacF1-infected strain had a growth rate and a growth amount reduced to about 2/3 as compared with the uninfected MAFF strain (hereinafter also simply referred to as “uninfected host strain”).

(Example 3: DNA isolation)
Isolation of DNA, cleavage with restriction enzymes and other nucleases, and construction of recombinant DNA were performed using standard molecular biology techniques as follows.

XacF1 phage DNA was isolated from the purified XacF1 phage particles by phenol extraction. In some cases, extrachromosomal DNA was isolated from bacteria infected with XacF1 phage by small-scale preparation with alkaline SDS solution. Replicate (RF) DNA for sequence analysis was isolated from host bacteria infected with XacF1 phage, treated with S1 nuclease, and shotgun sequencing was performed using Roche GS Junior Sequence System.

Next, assembly of the obtained array was performed using GS De Novo Assembler v2.6. The analyzed sequence corresponded to 156 times the final genome size (7325b) of the XacF1 phage. The nucleotide sequence data was analyzed with a computer using DNASIS v3.6 (manufactured by Hitachi Software Engineering). ORF candidates longer than 80b were identified using ORF Finder (http://www.ncbi.nlm.nih.gov/golf/golf.html). Sequence alignment was performed using the ClustalW program. To assign potential functions to the ORF, the DDBJ / EMBL / GenBank database was searched using the FASTA, FASTX, BLASTN and BLASTX programs.

(result)
The genome of XacF1 phage was circular single-stranded DNA consisting of 7325b. FIG. 3 shows a genetic map of the XacF1 phage compared to E. coli M13, which is a representative of Invivorus (Ff phage). The DNA replication module (R), structural protein module (S), and association / secretion module (AS) of XacF1 phage are modules common to M13. Unlike the M13, the XacF1 phage genome had a control region downstream of the association / secretion module. Details of the predicted ORF of the XacF1 phage are shown in Table 2.

(Example 4: Evaluation of motility of XacF1 phage-infected bacteria)
The assessment of Swiming motility and Swarming motility was as follows: 0.3% (w / v) and 0.7% (w / v) nutrient agar (Nutrient agar; Difco ™, Franklin Lakes, NJ, USA) Performed in The culture broth cultured overnight was centrifuged at 4 ° C. for 2 minutes (8000 × g), and the cells were washed with sterilized water and then suspended in sterilized water so that OD _{600 nm} = 1.0. 2 μL of the bacterial suspension was spotted on a petri dish having a diameter of 90 mm containing 20 mL of nutrient agar medium, and the bacterial migration range was measured while keeping the temperature at 28 ° C.

Twitching motility was evaluated as follows. As above, 2 μL of bacterial suspension was added to minimal nutrient medium (MM; 0.125% (NH ₄ ) ₂ SO ₄ , 0.175% K ₂ HPO ₄ , 0.075% KH ₂ PO ₄ , 0.025 % MgSO ₄ , 0.015% sodium citrate, 1.5% agar (w / v)). While culturing at 28 ° C., the periphery of the colony was observed with a microscope (100 times).

(result)
The results of the swimming motility are shown in FIGS. 4 (A) to 4 (C). Compared to the uninfected host strain shown in FIG. 4 (A), it was observed that the XacF1 phage-infected strain shown in FIG. 4 (B) did not spread on the medium. When the motility distance was quantified, as shown in FIG. 4 (C), it was shown that the XacF1 phage-infected strain had a lower swimming motility than the uninfected host strain.

FIGS. 5 (A) to (C) show the results of the warming motility. Similar to the swimming motility, it was observed that the XacF1 phage-infected strain shown in FIG. 5 (B) did not spread on the medium as compared to the uninfected host strain shown in FIG. 5 (A). When the motility distance was quantified, as shown in FIG. 5 (C), it was shown that the XacF1 phage-infected strain has a lower Warming motility than the uninfected host strain.

6 (A) and 6 (B) show the results of Switching motility. In the uninfected host strain shown in FIG. 6 (A), an irregular structure derived from Switching motility was observed around the colony. On the other hand, the shape around the colony of the XacF1 phage-infected strain shown in FIG. 6 (B) was smoother than that around the colony of the uninfected host strain. That is, it was shown that the Twitching motility of the XacF1 phage-infected strain is lower than that of the uninfected host strain.

(Example 5: Production evaluation of extracellular polysaccharide (xanthan))
The exopolysaccharide contained in the culture supernatants of the XacF1 phage-infected strain and the uninfected host strain was quantified by the following method. In NB medium supplemented with 2% (w / v) D-glucose, XacF1 phage-infected strain or uninfected host strain was cultured with shaking at 28 ° C. for 24 hours (200 rpm). Ten mL of the culture was centrifuged (5000 × g for 20 minutes) to remove the cells. The culture supernatant was mixed with 3 times the amount of 99% ethanol, cooled at 4 ° C. for 30 minutes, and centrifuged to collect extracellular polysaccharide as a precipitate. The recovered extracellular polysaccharide was dried at 55 ° C. overnight and quantified.

(result)
In the XacF1 phage-infected strain, 0.6 mg of exopolysaccharide was recovered from 10 mL of the culture solution. On the other hand, in the uninfected host strain, 6.7 mg of exopolysaccharide was recovered from 10 mL of the culture solution. The XacF1 phage-infected strain produces about 1/10 of the exopolysaccharide compared to the uninfected host strain, producing almost exopolysaccharide, particularly xanthan, which is observed in yellow as shown in FIG. It was shown not to. The exopolysaccharide produced by citrus canker is a virulence factor for citrus canker. For this reason, it was shown that the pathogenicity of the XacF1 phage-infected bacteria is greatly reduced.

(Example 6: Evaluation of pathogenicity of XacF1 phage-infected strain)
The pathogenicity of the XacF1 phage-infected strain was evaluated by the following method. After the lemon leaf was carefully washed with tap water, the leaf was completely spread, sterilized by soaking in sodium hypochlorite for 2 minutes, and washed with sterile water. Leaves were placed on the filter paper surface with the spine surface facing up. Each suspension of the XacF1 phage-infected strain and the uninfected host strain was inoculated into the leaves by a needle stick method and an osmotic method (10 ⁸ cfu / mL with sterile water). In the needle stab method, the leaves were stabbed with a needle, and 10 μL of each suspension was added dropwise to the puncture site. In the permeation method, the front side of the leaf was supported with a finger, and a syringe containing each suspension was pressure-inoculated on the back side. For observation, the treated area was marked directly after inoculation.

In the needle stab method and the penetrating method, inoculated leaves were covered with a plastic bag for 48 hours in order to promote infection. Leaves were placed in a 28 ° C. cultivation box for 4 weeks with a photoperiod of 12 hours light and 12 hours dark.

(result)
FIG. 8 shows the results of the needle stick method. In the uninfected host strain, lesions became prominent after 1 week, and the lesions expanded and raised over time, and typical symptom after 2 weeks. After that, Kaiyo further expanded. On the other hand, in the XacF1 phage-infected strain, clear lesions were hardly seen even after 4 weeks. The result of quantifying the lesion area in each of the 20 leaves is shown in FIG. The lesion caused by the XacF1 phage-infected strain hardly appeared, indicating that the XacF1 phage-infected strain was not pathogenic.

FIG. 10 shows the results of the infiltration method. The upper row shows the back side of the leaf, and the left two sites are inoculated with an uninfected host strain, and the right side is inoculated with a XacF1 phage-infected strain. The lower row shows the front side of the upper leaf. In the uninfected host strain, the inoculated site was decolored after 1 week, and clear symptom was observed. Two weeks later, the inoculation site turned brown and the tissue began to collapse. Four weeks later, further progress was made in tying. On the other hand, in the XacF1 phage-infected strain, no noticeable change was observed even after 3 weeks, and a slight yellowing boundary appeared around the inoculation site after 4 weeks. In the leaves inoculated with the XacF1 phage-infected strain, no symptoms of scab disease were observed. This result also indicates that the XacF1 phage-infected strain has no pathogenicity.

(Example 7: Preventing citrus canker disease of XacF1 phage)
The citrus canker prevention effect of the XacF1 phage-infected strain was evaluated by the following method. After the lemon leaf was carefully washed with tap water, the leaf was completely spread and sterilized by immersing it in a sodium hypochlorite solution for 2 minutes, followed by washing with sterile water. Leaves were placed on the filter paper surface with the spine surface facing up. A sterile water suspension (10 ⁸ cells / mL) of XacF1-infected strain (Xanthomonas pv. Citri MAFF301080) was sprayed onto the leaf surface with a nebulizer (1 mL / leaf). As a control, an uninfected host strain (MAFF301080) was sprayed on the leaf surface under the same conditions. After spraying, the leaves were stored in a petri dish adjusted for humidity at a photoperiod of 12 hours light period and 12 hours dark period at 28 ° C. for 4 weeks and observed.

(result)
As shown in FIG. 11 (A), in the lemon leaves sprayed with the uninfected host strain, the infection of the bacteria became clear after 3 weeks, and the infection spread in a wide leaf area after 4 weeks. On the other hand, as shown in FIG. 11 (B), in the leaves of the lemon sprayed with the XacF1-infected strain, no change was observed on the leaf surface even after 4 weeks. In addition, the same result was obtained in all leaves using five lemon leaves for each experiment.

(Example 8: Prevention effect of citrus canker disease of XacF1 phage-infected strain)
Lemon leaves that had been sprayed with XacF1 phage-infected strains were stabbed with a needle two days after spraying, and MAFF301080 wild strain sterilized water suspension (10 ⁸ cells / mL) was dropped into the stabbed area. (10 μL per location). As a control, the above-mentioned sterilized water suspension was similarly inoculated on a lemon leaf that was not sprayed. After inoculation, lemon leaves were stored and observed in the same manner as in Example 7 above.

(result)
In the absence of spray treatment, obvious symptom occurred after 2 weeks. As shown in FIG. 12 (A), browning and tissue collapse became prominent after 3 weeks. Four weeks later, the progression of symptoms was observed. On the other hand, as shown in FIG. 12 (B), the sprayed lemon leaves show some wounds on the inoculated portion after 3 weeks, but no symptoms are observed. There was no change after 4 weeks, and there was no symptom. In addition, the same result was obtained in all leaves using five lemon leaves for each experiment.

As shown in Example 8, the lemon leaves inoculated with the XacF1 phage-infected strain showed no signs of citrus scab even when exposed to wild strains of pathogenic citrus scab. Therefore, the XacF1 phage-infected strain is useful as a preventive agent for citrus canker disease.

The present invention is capable of various embodiments and modifications without departing from the broad spirit and scope of the present invention. The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications made within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2014-126782 filed on June 20, 2014. The specification, claims, and entire drawings of Japanese Patent Application No. 2014-126782 are incorporated herein by reference.

The present invention is suitable for prevention, expansion prevention, or control of citrus canker against fruit trees, mainly citrus. In particular, it is suitable for prevention and prevention of scabies in high-grade citrus fruits such as lemon and navel orange which are susceptible to scabies.

Claims

The structure of the phage particle is linear and infects citrus canker.
Bacteriophage.
The genome size is 7000-8000b,
The bacteriophage according to claim 1.
The genomic organization includes a DNA replication module, a structural protein module, an association / secretion module and a control region,
The bacteriophage according to claim 1 or 2.
The genomic base sequence consists of the base sequence shown in SEQ ID NO: 1.
The bacteriophage according to any one of claims 1 to 3.
The citrus canker can be obtained from Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. citri, or Xanthomonas citri,
The bacteriophage according to any one of claims 1 to 4.
A citrus canker prevention agent comprising the bacteriophage according to any one of claims 1 to 5.
A citrus canker prevention agent comprising a citrus canker disease fungus infected with the bacteriophage according to any one of claims 1 to 5.
The citrus canker can be obtained from Xanthomonas campestris pv. Citri MAFF301080 strain infected with the bacteriophage (name of depositary organization: National Institute of Technology and Evaluation, Patent Microorganism Depositary, Address of depositary organization: 2 Kazusa Kamashishi, Kisarazu City, Chiba Prefecture, Japan 292-0818 -5-8 Room 122, Deposit Date: June 16, 2014, Deposit Number: NITE BP-01875)
The citrus canker prevention agent according to claim 7.
Administering the citrus scab prevention agent according to any one of claims 6 to 8 to a plant or a plant growth medium.
How to prevent citrus canker disease.
The plant is a citrus.
The method for preventing citrus canker according to claim 9.