JP2016032435A - Bacteriophage, detection agent for citrus xanthomonas axonopodis, detection method for citrus xanthomonas axonopodis, control agent of xanthomonas campestris, and control method of xanthomonas campestris - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bacteriophage, a detection agent for citrus Xanthomonas axonopodis, a detection method for citrus Xanthomonas axonopodis, a control agent of Xanthomonas campestris, and a control method of Xanthomonas campestris that can be effectively used to various Xanthomonas campestris fungi.SOLUTION: For a bacteriophage, the structure of a phage particle includes a head potion and tail portion. The diameter of the head portion of the phage particle of the bacteriophage is 70-90 nm. The bacteriophage is infected with Xanthomonas campestris fungus.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a bacteriophage, a detection agent for citrus canker, a method for detecting citrus canker, a control agent for citrus canker, and a method for controlling citrus canker.

  In the export of Wenzhou mandarin oranges to the United States, quarantine of citrus canker disease by bacteriophage test is obligatory (43 agricultural administration B 345, 43 agricultural administration B 1901). One of the conditions for exporting fresh fruit of Wenzhou mandarin orange to the United States is that the result of the bacteriophage test does not determine that the test fruit had citrus canker.

  The bacteriophage test is performed by adding bacteriophages Cp1 and Cp2 specific for citrus scab disease bacteria to the assay solution, culturing, and counting lytic plaques (plaques) and comparing them with controls. When citrus canker is sensitive to Cp1 or Cp2, that is, when Cp1 or Cp2 is infected, citrus canker can be lysed. Therefore, the presence or absence of citrus canker on the test fruit can be determined by counting plaques.

  Although sensitivity to Cp1 and Cp2 of citrus canker varies depending on the strain (see Non-Patent Document 1), 97% or more of citrus canker disease is sensitive to at least one of Cp1 and Cp2.

Hiroshi Shiotani, “Citron radish fungus Xanthomonas citri subsp. Citri”, Microbial Genetic Resource Utilization Manual, National Institute of Agrobiological Sciences, December 25, 2010, Vol. 29, p. 1-11

  However, about 3% of citrus scab strains causing disease in the country are not sensitive to Cp1 and Cp2. For this reason, there is a risk of erroneous determination even if there is a citrus scab on the test fruit. Accordingly, there is a need for bacteriophages that are sensitive to a wide range of citrus canker.

  In addition, bacteriophage lyses citrus canker bacterium by infecting citrus canker as described above, so it is expected to be applied to safe control technology that replaces copper agents conventionally used for citrus canker. ing. If there is a citrus scab that is not sensitive to bacteriophages, the scope of application of the control technique using the bacteriophages is limited.

  The present invention has been made in view of the above circumstances, and can be effectively used for a wide range of citrus scabs, bacteriophages, citrus scabs detection methods, citrus scabs detection methods, citrus scabs control agents, and citrus scabs. It aims at providing the disease control method.

The bacteriophage according to the first aspect of the present invention is:
The structure of the phage particle includes a head and a tail,
The diameter of the head is 70-90 nm,
Infects citrus canker.

In this case, the length of the tail is
It may be 180 to 220 nm.

The bacteriophage according to the first aspect of the present invention is as follows.
Infects citrus canker disease strain MAFF311130,
It is good as well.

The bacteriophage according to the first aspect of the present invention is as follows.
CpX received on June 18, 2014 at NITE AP-01877 on the Patent Microorganism Depositary, National Institute of Technology and Evaluation, Japan
It is good as well.

The bacteriophage according to the first aspect of the present invention is as follows.
Genomic DNA is labeled,
It is good as well.

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

The method for detecting citrus canker disease according to the third aspect of the present invention comprises:
The detection agent for citrus canker disease according to the second aspect of the present invention is used.

In this case, the method for detecting citrus canker disease according to the third aspect of the present invention,
A mixing step of mixing the detection agent for citrus canker disease and a sample derived from a specimen;
A detection step of detecting citrus canker disease bacteria contained in the mixture obtained in the mixing step;
It is good also as including.

In the detection step,
Culturing the mixture, and detecting citrus canker disease contained in the mixture based on the plaque formed;
It is good as well.

In the detection step,
Culturing the mixture, and detecting citrus canker disease bacteria contained in the mixture based on the amount of growth of the cells,
It is good as well.

In addition, the detection agent for citrus canker disease
Bacteriophage according to the first aspect of the present invention, wherein the genomic DNA is labeled,
In the detection step,
Quantifying the labeled genomic DNA of the bacteriophage;
It is good as well.

In the detection step,
Quantifying bacteriophage adsorbed to citrus canker in the mixture,
It is good as well.

The control agent for citrus canker according to the fourth aspect of the present invention is:
The bacteriophage according to the first aspect of the present invention is included.

The method for controlling citrus canker according to the fifth aspect of the present invention comprises:
An administration step of administering the control agent for citrus canker according to the fourth 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, since the host range of bacteriophage is wide, it can be effectively used for a wide range of citrus canker.

It is a figure which shows the growth inhibitory effect of the citrus scab disease fungus infected with the bacteriophage which concerns on this invention. It is a figure which shows a band pattern when each DNA of bacteriophage, Cp1, and Cp2 which concerns on this invention is processed with a restriction enzyme. It is a figure which shows a band pattern when the DNA of the bacteriophage which concerns on this invention is processed with a restriction enzyme. It is a figure which shows an example of the shape of the bacteriophage which concerns on this invention.

  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 detection agent for citrus scab pathogen according to the present embodiment includes a bacteriophage that infects citrus scab pathogen. In the bacteriophage, the structure of the phage particle includes a head and a tail. Such a phage particle structure is also referred to as a head-tail structure, a siphovirus-like structure, or a siphoviridae type. Tail fiber (tail fiber) can be seen in the tail. The structure of the phage particles can be observed with an electron microscope by negatively staining the phage particles with sodium phosphotungstate.

  A characteristic of the phage particles of the bacteriophage is that the head has a diameter of 70 to 90 nm. The head diameter may be 75-85 nm or 78-82 nm, depending on the genome size of the bacteriophage. The bacteriophage CpX shown in the following examples has a head diameter of 80 nm. The shape of the CpX head is almost an icosahedron.

  Another feature of the bacteriophage phage particles is that the tail length is 180-220 nm. The length of the tail may be 190 to 210 nm or 195 to 205 nm. CpX has a tail length of about 200 nm.

  The diameter of the head of the phage particle and the length of the tail are specific among bacteriophages that infect citrus canker. For example, although the structure of the phage particle of Cp1 is siphoviridae type, the diameter of the head is about 60 ± 5 nm. The tail of the Cp1 phage particle is non-shrinkable, and the length of the tail is about 135 ± 10 nm. In the case of Cp2, the structure of the phage particle is a podoviridae type, which is different from the structure of the phage particle of the bacteriophage. The diameter of the head of the Cp2 phage particle is about 60 ± 5 nm. Further, the tail of the Cp2 phage particle is about 15 ± 5 nm, which is considerably shorter than the bacteriophage according to the present embodiment.

  The citrus scab disease fungus infected by the bacteriophage according to the present embodiment is a pathogenic bacterium that causes diseases on the fruits, leaves, branches, etc. of citrus fruits. 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.

  The host range of the bacteriophage according to the present embodiment is wider than Cp1 and Cp2. That is, the bacteriophage can also infect citrus scabs that Cp1 and Cp2 cannot infect. Examples of citrus scab disease strains to which the bacteriophage is infected include MAFF301077, MAFF301080, MAFF6733011 and MAFF6733013 capable of infecting Cp1, MAFF302102, MAFF673011, MAFF6733010, MAFF6733018 and MAFF6730301, etc. capable of infecting Cp2, and Cp1 and Cp2 MAFF311130 etc. which are not infected are mentioned. It should be noted that the examples given here are for illustrative purposes, and the host range of the bacteriophage according to the present embodiment is considered to be wider.

  More specifically, the bacteriophage according to the present embodiment is, for example, CpX received by the National Institute for Product Evaluation and Technology Patent Microbiology Deposit Center on June 18, 2014 with a receipt number: NITE AP-01877. It is.

  The bacteriophage can be obtained by washing and centrifuging a sample containing soil and filtering the supernatant with a membrane filter. Furthermore, the target bacteriophage can be isolated by using the above citrus scab pathogen suitable as a host. Bacteriophage isolation and titer measurement include a plaque assay in which an agar medium is overlaid with a soft agar medium (0.75% agar) in which a mixture of a test bacterium and a bacteriophage sample is added to form a plaque. Is preferred.

  As a 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 detection agent according to the present embodiment contains the bacteriophage in a state suspended in a solvent such as sterilized water or SM buffer. The detection agent may contain, for example, the bacteriophage at a concentration of 10 ² to 10 ¹⁰ pfu / mL, or 10 ⁴ to 10 ⁸ pfu / mL.

  The detection agent is suitable for use in a method for detecting citrus canker disease. By using the detection agent in, for example, a plaque assay, a wide variety of citrus canker can be detected. An example of a method for detecting citrus canker disease using the detection agent will be described below.

  The method for detecting citrus scab disease fungus comprises a mixing step of mixing the citrus scab disease fungus detection agent and the sample derived from the sample according to the present embodiment, and a detection for detecting citrus scab fungus contained in the mixture obtained in the mixing step. Steps. The specimen-derived sample is, for example, an assay solution in which a specimen such as a fruit is washed with sterilized gauze using sterilized gauze, and water remaining after washing is collected.

  In the mixing step, a detection agent is added to the assay solution. Here, for example, in order to concentrate bacteria in the assay solution, the assay solution may be centrifuged. In this case, a liquid medium such as a potato semi-synthetic liquid medium (PS medium) or NB medium is added to the precipitate obtained by centrifugation and suspended. An appropriate amount of the detection agent according to the present embodiment is added to the obtained suspension.

  Since the bacteriophage contained in the detection agent according to the present embodiment specifically infects citrus scab, it is detected in the detection step that the bacteriophage is infected with the bacteria contained in the mixture obtained in the mixing step. do it. For example, in the detection step, the mixture is cultured, and citrus canker disease contained in the mixture is detected based on the formed plaque. More specifically, after adding the detection agent to the liquid medium in which the precipitate is suspended as described above and mixing, the number of bacteriophages contained in the supernatant of the mixture recovered immediately by centrifugation, and 6-12 after mixing. This is confirmed by comparing the number of bacteriophages contained in the supernatant of the mixture incubated at 28 ° C. and then collected by centrifugation. When citrus canker that becomes a bacteriophage host is contained in the sample-derived mixed solution, the bacteriophage infects the host and proliferates during the culture for 6 to 12 hours. The number of increases. As a result, it is possible to detect citrus canker disease in the specimen.

  In the above detection step, a control obtained from a sample in a disease-free area is prepared, and when the number of plaques in the test solution is increased by a predetermined percentage or more, for example, 20% or more, compared to the number of plaques in the control, It is good also as a thing in which the citrus canker disease was detected by the test substance.

  Further, in the detection step, the mixture may be cultured, and citrus itch fungi contained in the mixture may be detected based on the amount of bacterial cell growth. The growth amount of the microbial cells after the culture can be measured, for example, by the absorbance of light having a wavelength of 600 nm that serves as an index of the bacterial concentration. Since the bacteriophage contained in the detection agent according to the present embodiment lyses the infected citrus canker, the amount of growth of the citrus canker decreases by lysis by the bacteriophage. Therefore, when the amount of the final cells when cultured with the detection agent added is smaller than when cultured without adding the detection agent, it can be said that the specimen contains citrus canker disease. In addition, for samples without citrus scab disease bacteria and samples with citrus scab disease fungus, a reference value for the amount of bacterial cells in a predetermined culture condition is obtained, and the amount of cultured cells related to the specimen is compared with the reference value. By doing so, it is possible to detect citrus canker disease in the specimen.

  As described above in detail, since the detection agent for citrus canker disease according to the present embodiment includes bacteriophages having a wide host range, it can accurately detect various citrus canker diseases.

  In addition, the bacteriophage contained in the detection agent for citrus canker disease according to the present embodiment includes citrus canker that does not infect Cp1 and Cp2 in addition to citrus canker disease that is infected with Cp1 and Cp2 used in the current bacteriophage test. It also infects the scab strain MAFF311130. For this reason, the accuracy of the bacteriophage test can be improved. Further, if the bacteriophage is used, the bacteriophage used in the bacteriophage test can be changed from two types of Cp1 and Cp2 to one type, so that the bacteriophage test can be simplified. As a result, it is possible to suppress as much as possible the dangers of operation mistakes, sample contamination, and the like caused by using two types of bacteriophages, and to efficiently detect citrus canker.

  Of course, when detecting citrus canker, the citrus canker detection agent according to this embodiment may be used in combination with Cp1 and Cp2. Since Cp1 and Cp2 have different strains of citrus canker, the presence of Cp1 and Cp2 can be confirmed (typing).

(Embodiment 2)
The detection agent for citrus canker disease according to the present embodiment includes the bacteriophage of the above-described embodiment 1 in which genomic DNA is labeled. Any label can be used as long as it can detect genomic DNA. However, a label that does not impair the function of the bacteriophage is preferable. The label is, for example, a fluorescent dye bound to a base of DNA, a gene encoding a fluorescent protein incorporated in the genome, or the like.

  Examples of the fluorescent dye include DAPI (4 ', 6-diamidino-2-phenylindole) or SYBR (trademark) Gold which binds to DNA. When the above-mentioned bacteriophage in which a fluorescent dye such as DAPI or SYBR ™ Gold is bound to genomic DNA is infected with citrus canker, the genome of the bacteriophage labeled with the fluorescent dye is transferred into citrus canker and under the fluorescence microscope. With fluorescence, fluorescence can be identified as an index. A fluorescent dye such as DAPI can be bound to genomic DNA by a known method. A gene encoding a fluorescent protein can be linked, for example, to a gene encoding a structural protein of bacteriophage. By doing so, the bacteriophage expressing the fluorescent protein can be identified. Examples of the gene encoding the fluorescent protein include a gene encoding green fluorescent protein (GFP). A gene encoding a fluorescent protein can be incorporated into genomic DNA by standard molecular biological techniques, genetic recombination techniques, and the like.

  A method for detecting citrus canker disease using the detection agent according to the present embodiment will be described below. In the detection method, the labeled genomic DNA of the bacteriophage is quantified in the detection step after the mixing step. In the case of labeling with a fluorescent dye as described above, citrus scabs that emit fluorescence observed under a fluorescence microscope may be counted in the detection step. Moreover, you may measure the intensity | strength of the fluorescence which a fluorescent pigment emits, a spectrum, polarized light, etc. using a fluorescence measuring apparatus. Similarly, when labeled with a fluorescent protein, the intensity of the fluorescent protein emitted by the fluorescent protein, spectrum, polarization, etc. are measured using a fluorescence measuring device or by counting the citrus scab that emits fluorescence observed under a fluorescence microscope. do it.

  As described above in detail, since the detection agent for citrus canker disease according to the present embodiment is labeled with genomic DNA, a wide variety of citrus canker disease bacteria can be detected more quickly and easily.

  The labeling of genomic DNA in this embodiment is not limited to fluorescent dyes and fluorescent proteins, and radioactive substances may be used.

(Embodiment 3)
As shown in Example 2 below, the bacteriophage contained in the detection agent according to the first embodiment or the second embodiment efficiently adsorbs to a wide range of citrus canker pathogens including MAFF311130 in which neither Cp1 nor Cp2 is infected. It has the characteristics of. Therefore, the method for detecting citrus canker disease according to the present embodiment utilizes the adsorptivity of bacteriophage to citrus canker disease. In the detection method, the bacteriophage adsorbed to citrus canker in the mixture is quantified in the detection step following the mixing step.

  In order to quantify the bacteriophage adsorbed on the citrus scab, it is only necessary to count the bacteriophage adsorbed on the surface layer of the citrus scab by using an electron microscope or the like. In this case, by using the detection agent containing the bacteriophage labeled with the fluorescent dye according to Embodiment 2, the bacteriophage fluorescent dye adsorbed on citrus canker can be easily counted under a fluorescence microscope.

  In addition, the bacteriophage adsorbed to citrus canker can be quantified by performing a plaque assay on a sample obtained by allowing the mixture obtained in the mixing step to stand for a predetermined time, centrifuging, and filtering the supernatant. In this case, plaques are formed depending on the number of bacteriophages that have not adsorbed to citrus canker. Here, as a control, for example, a diluted solution obtained by diluting bacteriophage with sterilized water instead of the assay solution is centrifuged, and a plaque assay is performed on the supernatant. Since the diluted solution does not contain citrus canker, the bacteriophage in the diluted solution does not adsorb to the citrus canker. For this reason, the bacteriophage contained in the supernatant is larger than the assay solution, and more plaques are formed. In this case, the difference between the number of control plaques and the number of plaques in the sample relative to the number of control plaques indicates the proportion of adsorbed bacteriophage. By calculating this ratio, the bacteriophage adsorbed on citrus canker can be quantified.

  As described in detail above, the method for detecting citrus canker disease according to the present embodiment can be detected at the stage where the bacteriophage is adsorbed without lysing the citrus canker disease, so that detection including the culture time and the like is possible. It is possible to reduce the time required for

  In addition, the said bacteriophage can be utilized also for removal, capture | acquisition, etc. of a citrus scab by utilizing the adsorptivity to a citrus scab. For example, bacteriophage is adsorbed to citrus scabs in a sample solution by passing the sample solution containing citrus scabs through a column packed with a carrier on which the bacteriophage is immobilized, and citrus scabs are captured on a carrier. Can do.

(Embodiment 4)
The bacteriophage according to the first embodiment has a wide host range as shown in Example 1 below, and lyses infected citrus canker. Therefore, the bacteriophage is suitable as an active ingredient of a control agent for citrus canker.

The control agent for citrus canker according to the present embodiment includes the bacteriophage according to the first embodiment. The control agent contains, for example, bacteriophage at 10 ³ to 10 ¹⁴ pfu / mL, 10 ⁴ to 10 ¹² pfu / mL, or 10 ³ to 10 ⁸ pfu / mL in a state of being suspended in sterilized water. In addition to bacteriophage, which is an active ingredient, the control agent may contain other substances, compositions, etc. that are generally pharmaceutically or botanically acceptable.

  The control agent for citrus canker according to the present embodiment can be used in a method for controlling citrus canker. The method for controlling citrus canker includes an administration step of administering the above-mentioned citrus canker control agent to a plant or a plant growth medium. The plant may be of any kind as long as it can be affected by 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 control agent for citrus canker is arbitrary as long as it can expose the plant or the plant growth medium to the control agent. The method of administering the control agent includes, for example, spraying and injecting a control agent for citrus canker, or allowing the control agent to penetrate into a plant or a plant growth medium. When injecting into a plant, the control agent may be put in a syringe and inoculated with pressure, or may be inoculated through an injection needle.

  If the plant is not infected with the pathogenic citrus scab fungus by administering the citrus scab disease control agent to the plant by spraying or the like, the plant is infected with the citrus scab or the disease of the citrus scab. 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 plant growth medium is a structure such as soil, mat, solid medium, nutrient solution in hydroponics, water in hydroponics, and the like. By administering the citrus scab control agent to the plant growth medium, the bacteriophage contained in the citrus scab control agent can be infected with a potential pathogenic citrus scab. As a result, citrus canker can be controlled. Here, “control” 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 dose of the bacteriophage contained in the citrus canker control agent according to the present embodiment is 10 ² to 10 ¹⁰ pfu, preferably 10 ⁴ to 10 ⁸ pfu per plant individual. The control agent for citrus canker includes the above-mentioned dose of bacteriophage in a suitable carrier or diluent, 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. is there. The control agent for citrus canker 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 depending on the concentration of bacteriophage, or in any amount higher than this. May be. When a suspension containing the citrus canker control 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, 1 to 5 mL are sprayed per 1 m ² of the surface area of the plant growth medium. Or may be administered in any amount beyond this.

  The citrus canker control agent according to the present embodiment may be administered once to a plant or a plant growth medium, or may be administered multiple times to a plant or a plant growth medium at an arbitrary time interval. For example, the control agent for citrus canker may be administered to plants or the like once or a week, or once every two weeks, once every three weeks, once every four weeks, or the like. The administration interval can be appropriately determined according to the plant to be administered or the plant growth medium.

  As described above in detail, the citrus canker disease control agent according to the present embodiment can lyse a wide variety of citrus canker diseases. For this reason, the said control agent of citrus canker can control citrus canker.

  Since the bacteriophage contained in the citrus canker disease control agent according to the present embodiment has a property of specifically infecting various citrus canker diseases, it has a high specificity and affects other useful microorganisms. Don't give. Thereby, the said control agent can make the influence on an environment as small as possible. Therefore, the control agent is safer than chemical pesticides, and can avoid problems such as an increase in resistant bacteria, an increase in effective application amount, environmental pollution, residual agricultural chemicals, and health effects.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples. Hereinafter, bacteriophage is also simply referred to as “phage”.

(Example 1: Isolation of bacteriophage and examination of host range)
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.

  Bacteriophage CpX was isolated from a soil sample collected from 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. (Plaque assay). Single plaques on NB plates were collected using a tip-tipped micropipette and 500 μL SM buffer (50 mM Tris / HCl pH 7.5, 100 mM NaCl, 10 mM MgSO ₄ and 0.01% gelatin). (W / v)). After vortexing, a plaque assay was performed using a filter sterilized with a membrane filter having a membrane pore diameter of 0.45 μm, and the CpX titer was determined.

CpX was grown on a small scale by the plate lysate method as follows. After performing a plaque assay under the condition that plaques can be formed on the entire surface of the NB plate (about 10 ³ to 10 ⁴ pfu), 6 mL of SM buffer was added to the plate. This was sealed with parafilm and cultured overnight at 4 ° C. with shaking. Plaque assay was performed using a filter sterilized supernatant with a membrane filter having a membrane pore diameter of 0.45 μm, and the titer was confirmed.

Growth on a large scale (100 mL to 1 L) was performed as follows. MAFF301077 was precultured in a test tube containing 4.5 mL of NB liquid medium (28 ° C., overnight, shaking culture). An appropriate amount from the pre-culture solution was added to 100 mL to 1 L of NB liquid medium for main culture (Difco ™, manufactured by BBL Becton Dickinson and Company, Cockeys Hill, MD, USA). When the MAFF301077 strain reached OD ₆₀₀ = 0.02 to 0.1, the suspension of CpX was added to M.P. O. The mixture was added so that I (multiplicity of infection) = 0.5 to 1, and allowed to stand at 28 ° C. for 30 minutes, and then cultured with shaking at 100 or 128 rpm for 6 to 8 hours. This culture solution was transferred to a 50 mL centrifuge plastic tube and centrifuged at 8000 rpm for 10 minutes using a cooling centrifuge (HITACHI) to precipitate cells. The supernatant was treated with DNase and RNase (10 μL each of DNase and RNase solutions were added to 50 mL of the supernatant and allowed to stand for 1 hour in a 30 ° C. hot water bath), and then the membrane filter with a membrane pore diameter of 0.45 μm Sterilized by filtration.

In order to examine the host range of CpX, the MAFF strain culture solution was adjusted to OD ₆₀₀ = 0.30, and 250 μL was placed in a 1.5 mL Eppendorf tube. An appropriate amount (10 to 100 μL) of CpX suspension was injected into this, and static culture was carried out at 28 ° C. for 20 minutes. The whole amount was added to Top agar (about 5 mL) and spread on an NB plate. Plaques were confirmed by static culture in a culture room at 28 ° C. for 1-2 days.

(result)
MAFF301077 was infected with CpX (MOI = 0.5 to 1.0), and cultured with shaking in NB liquid medium (28 ° C., 100 rpm). The time course of bacterial cell growth was compared with that of uninfected MAFF301077. As shown in FIG. 1, the cell growth rate and the final cell growth amount of the CpX-infected strain were greatly reduced as compared to the uninfected strain. For this reason, it was shown that CpX strongly suppresses the growth of citrus canker.

  Table 1 shows a portion of the CpX host range. “+” Indicates that CpX is infected, and “−” indicates that CpX is not infected.

  It was confirmed that CpX infects all MAFF strains including MAFF311130 in which Cp1 and Cp2 are not infected. In addition, although it investigated mainly on the pathogenic strain of lemon (Citrus limon) here, it is thought that the host range of CpX is still wider.

(Example 2: Evaluation of adsorption of CpX to citrus canker)
In the process of infection of the bacteriophage to the host, the bacteriophage is adsorbed on the host surface. For this reason, the adsorption specificity of bacteriophage is an important factor that determines the host range. Therefore, the adsorptivity of CpX to bacteria was evaluated.

A dilution series of CpX was prepared (10 ⁶ to 10 ³ pfu / mL), and 50 μL was dispensed into a 1.5 mL plastic test tube. 450 μL of the culture solution of the bacterium to be assayed prepared at OD = 0.1 was added to this (at 10 ⁶ pfu / mL, MOI = 0.001), and the mixture was allowed to stand at 28 ° C. for 20 minutes. Samples were centrifuged at 8000 rpm for 10 minutes at 4 ° C. and 200 μL of the supernatant was filter sterilized. About 10 μL of this supernatant, a plaque assay was carried out using MAFF301077, and the plaque number was counted after standing overnight at 28 ° C. (this is referred to as “actual measurement value”). Further, as a control, a diluted solution of CpX was centrifuged using ddH ₂ O instead of the assay culture solution, and a plaque assay was performed on 10 μL of supernatant using MAFF301077, and the number of plaques was counted ( This is called “theoretical value”).
The adsorption rate is
Adsorption rate (%) = 100 × (theoretical value−actually measured value) / theoretical value. In addition, XL10-GOLD was used as E. coli for comparison.

(result)
Table 2 shows the results of the above three tests and the average value. It was shown that CpX does not adsorb to E. coli but specifically adsorbs to the MAFF strain. Among the MAFF strains, it was shown that the adsorption rate to MAFF311130 in which Cp1 and Cp2 cannot be infected is extremely high.

(Example 3: Genomic extraction of CpX and characterization by genomic DNA)
In order to analyze the genomic DNA of CpX, first, phages were concentrated by PEG precipitation. An equal amount of 20% PEG solution (2M NaCl) was added to the CpX suspension, mixed well, and allowed to stand overnight at 4 ° C. Centrifugation was performed at 4 ° C. and 8000 rpm for 30 minutes in a cooling centrifuge, and the precipitate was suspended in the TE solution.

Proteinase K (final concentration: 1 mg / mL) and sodium N-lauroyl sarcosine (final concentration: 1%) are added to the concentrated phage suspension (about 10 ¹⁰ to 10 ¹² pfu / mL), and at 55 ° C., Incubated for 2 hours. To this was added an equal amount of phenol / chloroform / isoamyl alcohol (25: 24: 1), gently mixed for 5 minutes, and centrifuged at 4 ° C., 15,000 rpm for 15 minutes. Transfer the resulting supernatant to a new 1.5 mL plastic test tube, add an equal volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) and mix gently for 5 minutes, at 4 ° C., 15,000 rpm, Centrifuge for 15 minutes. A 10% amount of 3M sodium acetate and an equal amount of isopropyl alcohol were added to the supernatant and mixed gently. After standing at −50 ° C. for 1 hour, it is centrifuged at 4 ° C. and 15,000 rpm for 25 minutes, and the precipitate is rinsed with 70% ethanol and then vacuum-dried for 10 to 15 minutes to obtain an appropriate amount of TE solution or ddH. This was dissolved in ₂ O and used as a DNA sample.

(result)
The results of electrophoresis of a DNA sample treated with a restriction enzyme are shown in FIGS. FIG. 2 shows band patterns when Cp1, Cp2 and CpX were digested with HincII (lane a), HindIII (lane b), and PstI (lane c), respectively. Lane M is a lambda phage genomic DNA digested with StyI as a marker. The band pattern of CpX was clearly different from the band patterns of both Cp1 and Cp2, indicating that CpX is a different phage from Cp1 and Cp2. The genome size of CpX is about 50 kbp, which is slightly larger than Cp1 (43,870 bp) and Cp2 (42,963 bp).

  FIG. 3 shows bands obtained by digesting CpX with HincII (lane 1), EcoRV (lane 2), SacI (lane 3), DraI (lane 4), ClaI (lane 5), and XbaI (lane 6), respectively. Indicates a pattern. Lane M is obtained by digesting lambda phage genomic DNA with StyI as described above. From these band patterns, the genomic organization of CpX can be estimated.

(Example 4: Structure of phage particle of CpX)
CpX phage particles (10 ¹¹ pfu / mL) obtained using MAFF301077 as a host were negatively stained with phosphotungstic acid and observed with an electron microscope (Hitachi H600A). As shown in FIG. 4, CpX phage particles had a Siphovirus-like structure with a head diameter of 80 nm and a tail length of 200 nm.

  Various embodiments and modifications can be made to the present invention 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 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.

  INDUSTRIAL APPLICABILITY The present invention is suitable for controlling the scab of fruit trees, mainly citrus, for preventing the spread of scabs or for the control of citrus scab. In particular, it is suitable for the control and prevention of scab in high-grade citrus such as lemon and navel orange which are susceptible to scab.

Claims (15)

The structure of the phage particle includes a head and a tail,
The diameter of the head is 70-90 nm,
Infecting citrus canker,
Bacteriophage. The length of the tail is
180-220 nm,
The bacteriophage according to claim 1. Infects citrus canker disease strain MAFF311130,
The bacteriophage according to claim 1 or 2. CpX received on June 18, 2014 at NITE AP-01877 on the Patent Microorganism Depositary, National Institute of Technology and Evaluation, Japan
The bacteriophage according to any one of claims 1 to 3. Genomic DNA is labeled,
The bacteriophage according to any one of claims 1 to 4. Comprising the bacteriophage according to any one of claims 1 to 5,
A detection agent for citrus canker disease. Use of the detection agent for citrus scab pathogen according to claim 6.
A method for detecting citrus canker disease. A mixing step of mixing the detection agent for citrus canker disease and a sample derived from a specimen;
A detection step of detecting citrus canker disease bacteria contained in the mixture obtained in the mixing step;
The method for detecting citrus scab disease fungus according to claim 7 comprising: In the detection step,
Culturing the mixture, and detecting citrus canker disease contained in the mixture based on the plaque formed;
The method for detecting a citrus canker disease fungus according to claim 8. In the detection step,
Culturing the mixture, and detecting citrus canker disease bacteria contained in the mixture based on the amount of growth of the cells,
The method for detecting a citrus canker disease fungus according to claim 8. The detection agent for citrus canker disease
Bacteriophage according to claim 5,
In the detection step,
Quantifying the labeled genomic DNA of the bacteriophage;
The method for detecting a citrus canker disease fungus according to claim 8. In the detection step,
Quantifying bacteriophage adsorbed to citrus canker in the mixture,
The method for detecting a citrus canker disease fungus according to claim 8. Comprising the bacteriophage according to any one of claims 1 to 4,
A control agent for citrus canker. An administration step of administering the control agent for citrus canker according to claim 13 to a plant or a plant growth medium,
How to control citrus canker. The plant is a citrus.
The method for controlling citrus canker according to claim 14.