JPH03224482A - Novel carlavirus separated from japanese horseradish - Google Patents

Abstract

NEW MATERIAL:A virus or its mutant belonging to Carlavirus, separated from Japanese horseradish, exhibiting a string form of about (650-700)X13nm in size by direct negative method (DN method) and non-reactive with antiserum of turnip mosaic virus. USE:An antigen for the preparation of antibody for detecting infection with Carlavirus and a preventive and medicine for cochlearia virus disease. PREPARATION:The objective string-formed virus can be prepared e.g. by extracting shoot apex of Japanese horseradish, implanting and culturing the apex on an agar medium, proliferating a strain infected exclusively by Carlavirus by multiple shoot method, etc., chopping the obtained multiple shoot, putting into a buffer solution on an object glass to exude the juice, sucking excess liquid with a filter paper, spreading a collodion supporting film, drying in air and inspecting with a transmission-type electron microscope.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel virus belonging to Carlavirus isolated from wasabi husks that exhibits a degenerative phenomenon, and its mutant strain.

The virus of the present invention can be used as an antigen for producing antibodies to test whether wasabi is infected with these viruses that cause degeneration. Furthermore, it can also be used as a specimen for preventing or treating these wasabi virus diseases.

[Conventional technology]

Many varieties of wasabi (asabia 'a anica) are propagated by seedlings, but superior varieties such as ``Matsuma'' are propagated by division. However, by repeatedly dividing the plants, a so-called degeneration phenomenon occurs in which the rhizomes become poorly enlarged and almost no offspring can be produced, so the varieties of superior plants are on the verge of extinction.The main cause of this degeneration phenomenon is The cause is thought to be a viral disease caused by a viral infection [Haruo Suzuki et al., Shizuoka Agricultural Experiment Research Methods ■, 55
~66 (1976)).

Viruses isolated from wasabi include Monshien (Wasabi Tobacco Mosaic Virus), CMV (Chiewly Mosaic Virus), and TuMV (Turnip Mosaic Virus).
Three species have been reported. At present, no pesticide has yet been found that can kill viruses infecting plants and treat viral diseases. The shoot apical point culture method using tissue culture technology has been put into practical use for flowering plants such as orchids, yusas, and carnations, fruit trees such as grapes, apples, and tangerines, vegetables such as strawberries and yam, and medicinal plants such as nebula and japonica. has been done. The shoot apex point culture method has also been applied to wasabi, and its practical application is progressing using the Noricron method and the shoot primordium method.

However, not all seedlings produced by the shoot apical point culture method are virus-free, and virus testing of seedlings produced by the shoot apex point culture method is essential. Virus assay methods include bioassay methods, electron microscopy methods, and antiserum test methods, and it is necessary to perform assays using a combination of these methods depending on the type of plant and virus to be used as a control. As mentioned above, there are three types of viruses in wasabi, namely TMV-
Since it was known that the bacteria were infected only with Jl, CMV, and TuMV, tests to determine whether the cultured bacteria produced by the shoot apical culture method were virus-free were naturally conducted using only these three types of viruses as controls. Ta. However, as a result of testing, it has been discovered that even if the product is determined to be virus-free, it is actually infected with a virus.

[Problem to be solved by the invention]

The present invention attempts to search for and obtain a new virus that infects wasabi with the aim of ensuring virus testing of wasabi and obtaining wasabi that is completely virus-free. The resulting virus is intended to be used as an antigen for producing antibodies for wasabi virus assays and as a specimen for the prevention or treatment of these viral diseases.

[Means to solve the problem]

The present inventors conducted a search for a new virus in wasabi (variety: "Matsuma") obtained from a wasabi field in Yugashima Town, Shizuoka Prefecture, with the aim of obtaining a new virus for wasabi. As a result, three types of viruses were detected: rod-shaped viruses, string-shaped viruses, and rod-shaped to bullet-shaped viruses. Of these, the rod-shaped virus is 300nm x 18n■
Because it reacted well with the TMV-ga antiserum, it was identified as the wasabi type of the known tobacco mosaic virus. Regarding the string-shaped virus and the rod-shaped to bullet-shaped virus, as a result of the assay described below, it was confirmed that the virus is a new type of virus that has not been described in any literature, leading to the present invention.

The present invention relates to a new virus that belongs to the string-shaped virus Carlavirus among the two types of viruses mentioned above.

In addition, the Carla virus of the present invention is
High temperature treatment for about 15 days, chemical treatment with nitrous acid and hydroxyamine, and in vitro +*uta
gen-esis-reverse genetics
Although it can be mutated through genetic manipulation, etc.,
The present invention also encompasses such mutant strains.

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

a) Harvesting and Detection of Virus Isolation and propagation of Carlavirus were carried out by infecting Carlavirus alone using the shoot apex culture method, and infecting Melicurone alone. In other words, wasabi from Yuke, Izu (variety:
``Matsuma'') was used, 7 to 8 shoot tips per rhizome were removed, and the shoot tips were placed on a Murashigeskoog agar medium containing 1 μ/l of benzylaminopurine and 10 g/l of agar and cultured. . Cultivation was carried out at 18°C with irradiation for 116 hours/day.
The test was carried out in a 2000 lux artificial weather machine. Culture 1~
After 2 months, shoots that had grown to 1-2C1 were tested for viruses by electron microscopy, bioassay, and ELISA, and were found to be virus-free. lV mono-infected strain, Carlavirus mono-infected strain and TM mono-infected strain -
A mono-infected strain of P. lavirus was obtained and a mono-infected strain of P. arla
Carlavirus was propagated by multiplying a virus-infected strain by a multi-infection method. 0.1*oj2/41 on the slide glass! A drop of phosphate buffer was added, and the multiple shoots obtained above were cut into pieces and placed therein to allow the juice to ooze out.

Next, a collodion support membrane was applied, and the membrane surface of the carbon-reinforced copper grid was brought into contact with this wasabi juice liquid, and the excess liquid was immediately absorbed with a filter paper, and the membrane was air-dried with the membrane side facing up. Place a few drops of a fixative solution containing 1% glutaraldehyde in 0.1vxo1/1 phosphate buffer on a slide glass, and float an air-dried grid on the surface of the solution for 3 to 5 minutes with the sample side facing down. It was fixed. After washing the grid with the fixed sample three times with deionized distilled water, staining solution (2% phosphotungstic acid aqueous solution, dry well 0.5
% addition (pH 7.0) for 30 seconds, the excess staining solution was absorbed with a filter paper, and the membrane was air-dried with the membrane side facing up to prepare a sample for detection.

b) Observation of the morphology of the virus The sample thus prepared was examined using a transmission electron microscope to observe the morphology of the virus.

As a result, rod-shaped viruses, string-shaped viruses, and rod-shaped ~
Three types of bullet-shaped viruses were detected.

The rod-shaped virus has a size of 300nm x 18r+m, and is similar to TMV-H antiserum (known from the reaction with TMV-H antiserum).
It was identified as MV-W. TuMV is a string-shaped virus that has been reported to infect wasabi.
While the size of TuMV is 750 rho x 1 ltv, the string-shaped virus detected by the present invention has a shape as shown in Figure 1, and its size is 650 to 700 nm.
No pinwheel-shaped inclusion bodies were found, which are significantly different from X 13n+* and which are specifically found in plants infected with TuMV.

Furthermore, it did not react with TuMV antiserum at all. Based on the above, it was determined that the string-shaped virus of the present invention belongs to the Carlavirus family, which is different from TuMV. There are 2 rod-shaped to bullet-shaped viruses.
30-250nm x 85-90nm with 4.5 inside
This virus is classified as a rhabdovirus (Pl
It was determined that the virus belonged to the genus Anthrhabdovirus.

C) Intracellular findings of viruses using ultrathin section method A wasabi sample fixed by double fixation using glutaraldehyde and osmic acid was embedded and solidified in epoxy resin, and then ultrathin sections were prepared. The ultrathin sections were mounted on copper grids and stained using a double staining method with uranium acetate and lead citrate. The samples prepared in this manner were examined using a transmission electron microscope to observe the intracellular presence of the virus and changes in virus-infected cells.

The string-like virus belonging to the Carlavirus of the present invention is found scattered or clustered in the cytoplasm when observed using ultrathin sectioning, and the proliferation of membranous structures is sometimes observed in the cytoplasm of infected cells. Taga, TuM
No tabular or pinwheel-shaped cytoplasmic inclusions as seen in V-infected cells were detected.

d) Host range To determine whether the Carlavirus of the present invention infects other plants, an inoculation test was conducted on 13 species of plants from 2 families, including Chinese cabbage, shown in Table 1.

That is, wasabi fliclon infected only with Carlavirus was prepared, and the sap was inoculated to various plants using this as an inoculum. The virus test method involves observing disease symptoms on inoculated leaves and upper leaves, and
This was done using the N method.

Table 1 shows the test results of sap inoculation of 13 species of plants of 2 families. As can be seen from the results, no infection with Carlavirus was observed in any of the plants.

Chinese cabbage Chinese cabbage cabbage cauliflower broccoli 15 0/2 Kale O/2 Japanese radish (Minowase) 015 (red circle) 015 Santo rhinoceros 015 15 15 0/1 0/
11) Number of detected strains/number of tested strains Next, Carlavirus, rhabdovirus (PR, ant rhabdovirus)
and TMV-, the parent wasabi ``Matsuma'' (
Cultivar name) was used as an inoculum source to inoculate 6 families and 2, mainly from Brassicaceae plants.
Nine plants were inoculated with sap. Tables 2 and 3 show the results of sap inoculation to 29 species of plants in 6 families, mainly those of the Brassicaceae family. No infection with Carlavirus or Plant rhabdovirus was observed in any of the plants.

Carlavirus infects cruciferous plants
Only one type of Kale latin virus has been reported in Brazil, but as shown in Tables 1 and 2, the Carla virus discovered in the present invention does not infect kale, and this This revealed that the virus of the present invention is a new type of virus.

Table 2: Cabbage” Ego 11 Danya - Table 3: Tobacco (Xanthi-nc) (Sumsun) Nicotiana
No.) Red nfloridana Cucumber (art book) ChenO0diu-amaranticolor rainbow
Group L Cowpea (14 isconsin Perfection) (Top crop) Broad bean (-size) Ento (Usui)
(Effected Waies) e) Local outbreak investigation of new virus We conducted a virus outbreak investigation at wasabi cultivation sites.

Virus testing was conducted using the DN method on 31 samples of 5 wasabi varieties. Carravirus (Carravirus) discovered in the present invention
lavirus) was detected in specimens of five varieties, and it became clear that it is a very important virus for wasabi.

(Effects of the Invention) The present invention provides a new type of virus belonging to Carlavirus, which is isolated from wasabi that has undergone a degeneration phenomenon.

The new virus of the present invention is an antigen for producing antibodies for testing whether wasabi being cultivated is infected with this virus or for testing whether a culture produced by tissue culture is virus-free. It can be used as It can also be used as a specimen for research into the treatment or prevention of infection of wasabi infected with this virus.

As a result, deterioration of wasabi can be prevented and the quality of wasabi can be improved.

[Brief explanation of drawings]

FIG. 1 shows the microorganism carravirus (Car-1) of the present invention.
avirus) (magnification: 55,000 times).

Claims (1)

[Claims] (1) Separated from wasabi using direct negative method (
It showed a string-like shape of about 650 to 700 x 13 nm using the DN method), and it was similar to the turnip mosaic virus (turnip mosaic virus).
Virus belonging to Carlavirus (Carlavirus) or its mutant strain that does not react with the antiserum of Carlavirus (Saicvirus)