JPH0636736B2 - New Carlavirus isolated from horseradish - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel virus belonging to Carla virus isolated from horseradish showing degeneration and a mutant strain thereof.

The virus of the present invention can be used as an antigen for producing an antibody for assaying whether or not horseradish is infected with these viruses that cause the degeneration phenomenon. Further, it can be used as a sample for preventing or treating these wasabi virus diseases.

[Conventional technology]

Many varieties of wasabi (Wasabia japanica) are cultivated in seedlings, but excellent varieties such as "Matsuma" are cultivated in separate stocks. However, by repeating the stock splitting, a so-called degeneration phenomenon occurs in which the rhizome grows worse and the offspring can hardly be harvested. Therefore, the excellent stock splitting variety is endangered. It is considered that the main cause of this degeneration phenomenon is a viral disease caused by viral infection [Haruo Suzuki et al., Shizuoka Agricultural Res. Inst., 21 , 55-66 (1976)]. Virus is separated from wasabi TMV-W (Tobacco Mosaic Virus horseradish system) CMV (cucumber mosaic virus) and TuMV 3 species (turnip mosaic virus) have been reported [above掲誌, Yasuo Komuro et al, Plant Protection 20 , 486 ~ 488
(1966) Toshihara Hiroshi et al., Kanto Higashiyama Disease Research Bulletin 11 , 46 (196
Four)〕. To date, no pesticides have been found to kill viruses that infect plants and treat viral diseases. The shoot apex growth point culture method using tissue culture technology is almost the only method for removing so-called virus-free virus from plants infected with virus.
It has been put to practical use in flowering plants such as yusa and carnations, fruit trees such as grapes, apples and mandarins, vegetables such as strawberries and yams, and medicinal plants such as senkyu and dio. The shoot apical growth point culture method is also applied to horseradish, and its practical application is being promoted by the melicron method and the shoot primordium method.

However, not all the seedlings produced by the shoot apical point culture method are virus-free, and a virus assay for seedlings produced by the shoot apical point culture method is essential. Viral assay methods include bioassay methods, electron microscopy methods, and antiserum test methods, and it is necessary to combine these methods depending on the type of plant and virus used as controls. In wasabi, there are three types of viruses as mentioned above, namely TMV-W CM.
Since it has been known that only V and TuMV are infected, the test whether the cultured seedlings produced by the shoot apical culture method are virus-free or not has naturally been carried out using only these three kinds of viruses as controls. However, even if it is determined that the virus is free as a result of the test, it is actually found to be infected with the virus.

[Problems to be Solved by the Invention]

The present invention is intended to secure the virus assay of wasabi and to search for and acquire a novel virus that infects wasabi for the purpose of obtaining completely virus-free wasabi. The obtained virus is intended to be used as an antigen for producing an antibody for performing a horseradish virus assay and as a sample for preventing or treating these viral diseases.

[Means for Solving the Problems]

The present inventors searched for a new virus of wasabi (variety: "Matsuma") obtained from the wasabi field in Yugashima-cho, Shizuoka Prefecture for the purpose of obtaining a new virus of wasabi. As a result, three types of viruses, rod-shaped virus, string-shaped virus, and rod-shaped to bullet-shaped virus were detected.
Among them, the rod-shaped virus is TMV-W with a size of 300 nm × 18 nm.
It reacts well with the antiserum of
It was identified as the wasabi line of rus. As a result of an assay as described later, it was confirmed that the string virus and the rod-shaped to bullet-shaped virus were new species of viruses that have not been written yet, and the present invention has been completed.

The present invention relates to a novel virus belonging to Carla virus, which is a cord-shaped virus among the above-mentioned two viruses.

In addition, the carla virus of the present invention is exposed to high temperature treatment at 35 to 37 ° C. for about 5 to 15 days, chemical treatment with nitrite and hydroxyamine, and in vitro mutagen-esis-reverse geneti.
It can be mutated by gene manipulation by the cs method or the like, and such mutant strains are also included in the present invention.

The collection method and morphology of the virus of the present invention are as follows.

a) Virus collection and detection Carlavirus isolation and growth is carried out by the shoot apical method.
Single-infection meliclone was prepared and propagated by the multiple shoot method. In other words, the wasabi from Izu Yuga (variety: "Matsuma") was tested,
Seven to eight shoot tips were extracted per rhizome, and the shoot tips were placed on a Murashigeskoog agar medium containing benzylaminopurine 1 mg / and agar 10 g / and cultured. Culture is 18
It was carried out in an artificial weather device at 2000 lux for 16 hours irradiation at ℃. After 1-2 months of culture, shoots that had grown to 1-2 cm were virus-tested by electron microscopy, bioassay, and ELISA. Virus-free strain, TMV single-infected strain, Carlavirus single-infected strain and TMV.Carlavirus duplication Since an infected strain was obtained, Carlavirus was propagated by multiplying the Carlavirus-only infected strain by the multiple shoot method. A drop of 0.1 mo / phosphate buffer was dropped on a slide glass, and the multiple shoot obtained above was cut into the slide glass to exude the juice.

Next, a collodion support film was attached, and the film surface of a carbon grid reinforced copper grid was brought into contact with this wasabi juice liquid, and the excess liquid was quickly absorbed by a filter paper and air-dried with the film surface facing upward. Place a few drops of fixative solution of 1% glutaraldehyde in 0.1mo / phosphate buffer solution on a slide glass, float the sample surface of the air-dried grid on the liquid surface for 3-5 minutes, and fix it. It was The fixed sample grid was washed three times with deionized distilled water and then stained (2% phosphotungstic acid aqueous solution, 0.5% drywell).
The sample was dyed for 30 seconds with an added pH of 7.0), the excess dyeing solution was absorbed with filter paper, and air-dried with the film surface facing upward to obtain a sample for detection.

b) Observation of virus morphology The sample thus prepared was observed under a transmission electron microscope to observe virus morphology.

As a result, rod-shaped virus, string-shaped virus and bacillus-
Three bullet-like viruses were detected. The rod-shaped virus
It was identified as a known TMV-W because it had a size of 300 nm × 18 nm and reacted well with the antiserum of TMV-W. There is TuMV as a cord-shaped virus that has been reported to be infected with horseradish, whereas the size of TuMV is 750 nm × 11 nm.
The cord-like virus detected in the present invention has a morphology as shown in FIG. 1 and its size is significantly different from 650 to 700 nm × 13 nm, and a windmill-shaped inclusion body specifically found in a plant infected with TuMV. Was not found. Furthermore, it did not react with TuMV antiserum at all. From the above, the cord-like virus of the present invention is different from TuMV in Carla virus
It was determined that the virus belongs to. Since the rod-shaped to bullet-shaped virus has a nucleocapsid with a helical structure of about 4.5 nm at about 230 to 250 nm × 85 to 90 nm and a coating on the outside, this virus is a rhabdovirus (Pant virus).
rhabdovirus).

c) Intracellular observation of virus by ultra-thin section method An ultra-thin section was prepared after embedding and solidifying a horseradish sample fixed by the double fixation method with glutaraldehyde and osmic acid in epoxy resin. This ultrathin section was placed on a copper grid and stained by a double staining method with uranium acetate and lead citrate. The sample thus prepared was examined under a transmission electron microscope to observe the intracellular presence of virus and changes in virus-infected cells.

The cord-like virus belonging to the Carla virus of the present invention is found scattered or aggregated in the cytoplasm by observation by ultrathin section method, and sometimes the proliferation of membranous structures is observed in the cytoplasm of infected cells. However, no tobacco-like or windmill-like cytoplasmic inclusions found in TuMV-infected cells were detected.

d) Range of Hosts Chinese cabbage, etc. 2 family 13 shown in Table 1 as to whether the carlavirus of the present invention infects other plants
Inoculation tests were performed on seed plants.

In other words, we created wasabi meliclone that is only infected with Carla virus, and using this as the inoculum source,
Juice was inoculated into various plants. The virus assay was performed by observing the symptom on the inoculated leaf and the upper leaf and by the DN method.

Table 1 shows the results of the assay of sap inoculation on 13 species of plants from 2 families. As can be seen from these results, none of the plants was infected with Carla virus.

Next, Carlavirus, Rhabdovirus (P
ant rhabdovirus) and TMV-W mixedly infected with parental horseradish "Matsuma" (cultivar name) as a source of inoculation to 29 plants of 6 families, mainly Brassicaceae. Tables 2 and 3 show the results of sap inoculation of 29 plants of 6 families centering on such cruciferous plants. Infection of neither plant with Carla virus nor Rhabdovirus was observed. Carla virus infecting cruciferous plants
As Carla virus, only one kind of Kale Latent virus has been reported in Brazil, but the Carla virus found in the present invention does not infect Kale as shown in Tables 1 and 2, From this, it became clear that the virus of the present invention is a novel virus.

e) Local outbreak survey of new virus A virus outbreak in the wasabi cultivation field was conducted. A virus test was carried out by the DN method on 31 samples of 5 varieties of wasabi. Carlavirus found in the present invention
Was detected in samples of 5 varieties, and it became clear that it is a very important virus for horseradish.

〔The invention's effect〕

The present invention provides a new type of virus belonging to Carla virus isolated from horseradish in which degeneration has appeared.

The novel virus of the present invention is an antibody-producing antigen for assaying whether or not wasabi during cultivation is infected with this virus, or assaying whether or not the culture produced by tissue culture is virus-free. Can be used as In addition, it can be used as a sample of research for treatment or infection prevention of horseradish infected with this virus.

As a result, it is possible to prevent the degeneration phenomenon of the wasabi and improve the quality of the wasabi.

[Brief description of drawings]

FIG. 1 shows the microbial carlavirus of the present invention.
2 is an electron micrograph showing the morphology (magnification: 55,000 times).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shuichi Yamashita 1-3-16-104 Etchujima, Koto-ku, Tokyo (72) Inventor Tsuneo Tsuchizaki 43-14 Shiroyama, Kukizaki-cho, Inashiki-gun, Ibaraki Prefecture

Claims (1)

[Claims] 1. A cord-like morphology of about 650 to 700 × 13 nm separated from wasabi by direct negative method (DN method),
Virus belonging to Carla virus (Carla virus) that does not react with antiserum of Turnip mosaic virus (muta strain)