JPH0636735B2 - A novel rhabdovirus 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 the rhabdovirus (Pant rhabdovirus) isolated from horseradish in which a degeneration phenomenon appears, 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 japonica) are cultivated in seedlings, while excellent varieties such as "Matsuma" are cultivated by stocking. However, because the so-called degeneration phenomenon, in which the rhizome grows worse and the offspring can hardly be collected by repeating the stock splitting, the excellent stock split varieties are endangered. The main cause of this degeneration phenomenon is considered to be a viral disease due to viral infection. [Suzuki Haru et al., Shizuoka Agricultural Exploration Method 21 , 55-66 (1976)].
Three types of viruses isolated from wasabi have been reported: TMV-W (tobacco mosaic virus wasabi system) CMV (cucumber mosaic virus) and TuMV (turnip mosaic virus) [above journal, Yasuo Komuro et al., Plant protection 20 , 486 ~ 48
8 (1966), Hiroshi Tochihara et al., Kanto Higashiyama disease and bug research report 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 the only method for removing so-called virus-free virus from plants infected with virus.
It has been put to practical use in flowers such as lilies and carnations, fruit trees such as grapes, apples and mandarins, vegetables such as strawberries and yams, and medicinal plants such as syringa 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 horseradish, as mentioned above, there are three types of viruses, namely TMV-W.
Since it has been known that only CMV 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, as a result of the test, it is found that the virus is actually infected even if it is determined to be virus-free.

[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 virus thus obtained is intended to be used as an antigen for producing an antibody for performing a horseradish virus assay and as a specimen for preventing or treating these viral diseases.

[Means for Solving the Problems]

The present inventors searched for a new virus of wasabi (variety: Mazuma) obtained from the wasabi field in Yugashima Town, Shizuoka Prefecture, with the aim 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 had a size of 300 nm × 18 nm and reacted well with the antiserum of TMV-W, so that the known tabaco mosaic viru
s wasabi strain. As a result of an assay as described below, the cord-shaped virus and the bacillus-shaped to bullet-shaped virus were confirmed to be novel viruses that have not been written yet, and the present invention has been completed.

The present invention provides a rhabdo virus (Pant rhabdoviru) which is a rod-shaped to bullet-shaped virus among the above-mentioned two kinds of viruses.
s) relating to new viruses.

In addition, the rhabdovirus of the present invention is exposed to high temperature at 35 to 37 ° C. for about 5 to 15 days, chemical treatment with nitrite and hydroxyamine, and in vitro mutagen-esis-reverse geneti.
The present invention also includes such mutant strains, which can be mutated by genetic manipulation such as the cs method.

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

a) Virus collection and detection Wasabi obtained from the Wasabi field in Yugashima Town, Shizuoka Prefecture (variety:
"Matsuma") was chopped into 10 mm x 1 mm pieces, and further chopped. A drop of 0.1 mo / phosphate buffer was dropped on a slide glass, and the above chopped wasabi was put therein 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 rod-shaped virus ~
Three types of bullet virus were detected. The rod-shaped virus had a size of 300 nm × 18 nm and reacted well with the antiserum of TMV-W, so it was identified as a known TMV-W. There is TuMV as a cord-shaped virus that has been reported to be infected with horseradish, but the size of the cord-shaped virus detected is 650 to 700 nm, while the size of TuMV is 750 nm × 11 nm.
Unlike the x13 nm, no windmill-like inclusion bodies found in TuMV-infected plants were found. Furthermore, it did not react with TuMV antiserum at all. Based on the above, the cord-like virus is different from TuMV in terms of carla virus (C
arla virus). on the other hand,
The bacillus-bullet shaped virus of the present invention is about 230-250 nm × 85.
This virus was judged to belong to the rhabdovirus (Pant rhabdovirus) because it has a nucleocapsid with a helical structure of about 4.5 nm at ˜90 nm and a coat on the outside.

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 rod-shaped to bullet-shaped virus belonging to the rhabdovirus of the present invention was found in the nucleus, internuclear space and cytoplasm. Uncoated nucleocapsids were found in the nucleus along with virus particles. From the above findings, this virus was found to be a nuclear-proliferating virus.

d) Scope of Host Inoculation test was conducted on 29 species of 6 families, mainly Brassicaceae plants, to determine whether or not the rhabdovirus of the present invention infects other plants.

That is, rhabdovirus
Using the parent horseradish "Mazuma" (variety name), which is mixedly infected with Carla virus and TMV-W, as a source of inoculation, sap was inoculated into 29 species of 6 families, mainly Brassicaceae. The results are shown in Tables 1 and 2. Neither the rhododo (P-ant rhabdovirus) nor the carlavirus (Carlavirus) was infected to any of the plants. Infection of cruciferous plants with rhabdovirus (P-ant rhabdovirus) has been reported in Europe, broccoli necrotic yellow virus and Brazil, only two species from Raphanus sp. Have been reported. Virus (Pant rhabdovi
As shown in Table 1, rus) did not infect broccoli or Raphanus (radish), which revealed that this virus is a novel virus.

e) Local outbreak survey of new virus A virus outbreak in the wasabi cultivation field was conducted. That is, when 31 samples of 5 varieties of wasabi were tested by the DN method, rhabdovirus (Pant rh-abdoviru
s) was detected only in "Matsuma", and it became clear that this virus has an important influence on this wasabi cultivar in which the decrease in yield due to the degeneration phenomenon is a problem.

〔The invention's effect〕

The present invention provides a new kind of virus belonging to the rhabdovirus (Pant rhabdovirus) isolated from horseradish in which degeneration appears. 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 rhabdovirus of the present invention (Pant).
2 is an electron micrograph showing the morphology of rhabdovirus).
(Magnification 220,000 times)

(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 bacillus-like to bullet-like form of about 230 to 250 × 85 to 90 nm separated from horseradish by the direct negative method (DN method), which has a nucleocapsid with a helical structure inside and a coating on the outside. Virus belonging to Rhabdovirus or its mutants