JPS61177985A - Production of cucumber mosaic viral attenuated virus using sattelite rna and novel attenuated virus obtained therefrom and method for controlling cucumber mosaic virus using same - Google Patents

Abstract

PURPOSE:To obtain an attenuated virus of cucumber mosaic virus in high probability, by incorporating a sattelite virus obtained from cucumber mosaic viruses without exhibiting necrotic symptoms in tomatoes. CONSTITUTION:An attenuated virus of cucumber mosaic viruses obtained by incorporating a sattelite RNA, having 0.5-2.5X10<5> daltons molecular weight, and obtained from cucumber mosaic viruses without exhibiting necrotic symptoms in tomatoes. The above-mentioned attenuated virus is prepared by inoculating a sattelite RNA, having 0.5-2.5X10<5> daltons molecular weight, and obtained from cucumber mosaic viruses containing no cucumber mosaic virus and cucumber mosaic viruses without exhibiting necrotic symptoms in vegetables, flowering plants, tobacco and beans, and preparing the aimed attenuated virus from the sap of the vegetables, flowering plants, tobacco and beans.

Description

[Detailed description of the invention] [Industrial application field]

The present invention provides a method for producing a less virulent virus strain (hereinafter referred to as an attenuated virus) of cucumber mosaic virus (hereinafter abbreviated as CMV) that causes disease damage to vegetables, flowers, tobacco, and legumes, and a method for producing a less virulent virus strain (hereinafter referred to as an attenuated virus), and The present invention relates to a new attenuated CMV virus and a method for controlling CMV using the same.

[Conventional technology]

The attenuated tobacco mosaic virus (DingMV) is used to control TMV in tomatoes. This attenuated virus was created by selecting three isolates from heat-treated infected plants. However, there is currently no control technology for CMV that uses a weakened virus. CMV can be controlled by spraying pesticides to exterminate vector insects (aphids), preventing them from flying, and cultivating resistant varieties. There are also problems in terms of environmental pollution, and furthermore, the pest control effect is not sufficient. There are ways to prevent insect vectors from flying around, such as covering the soil with silver film or cheesecloth, but these methods are expensive, so their use is limited. In addition, there are almost no effective resistant varieties against CMv, and it takes many years to develop resistant varieties, and there are very few gene sources that can actually be used.

[Problems to be solved by the invention]

All of the above-mentioned methods have insufficient control effects against diseases caused by CMV, and there are problems such as economic efficiency and environmental pollution, so they cannot be used for various vegetables and flowers. Control of CMV, which occurs in tobacco and legumes, has hardly been carried out so far. Therefore, a control method using attenuated viruses can be considered as a method that is highly effective, economical, and free from environmental pollution. On the other hand, there are methods to create weak viruses, such as isolating natural viruses and treating them with heat or sodium nitrite, but both require the work of searching for weakened viruses from a large number of virus strains. , the probability of obtaining a weakened virus is extremely low. −

[Purpose of the invention]

The purpose of the present invention is to find an effective attenuated virus, a method for producing the same, and a method for using the same by investigating the component (satellite RNA) that controls the attenuation of CMV.

[Means to solve the problem]

The present inventors focused on the fact that among the nucleic acid components that constitute CMV, satellite RNA controls the internal expression of infected plants, and as a result of various studies, the nucleic acid components that control the severity of disease. By discovering that this is also a satellite RNA, we have completed the present invention. That is, the novel attenuated CMV virus of the present invention is a molecule obtained from CMV that does not cause erythema symptoms in tomatoes [10,
The CMV is characterized by having a satellite RNA of 5 to 2,5 x 10' daltons incorporated therein, and the method for producing such an attenuated CMV virus according to the present invention can be applied to a CMV that does not contain a satellite RNA and a CMV that does not exhibit symptoms of paralysis. Molecule II O, 5-2.5x 10' dalton satellite RNA obtained from vegetables. After inoculating flowers, tobacco, and beans, the vegetables,
The CMV? according to the present invention is characterized by producing attenuated viruses from the sap of flowers, tobacco, and beans. 11J! The control method using a virus is characterized by inoculating the novel CM V 5v3m virus into seedlings of target plants in advance.

[Example] Hereinafter, the present invention will be explained in more detail with reference to the drawings. The nucleic acid of CMV consists of RNA, but when this viral nucleic acid is separated by electrophoresis using polyacrylamide gel (501A/gel, 4-5 hours), as shown in Figure 1, various CMVs are composed of four components of RNA (RNA1 to RNA5) and five component RNAs (RNA1 to RNA5)
It can be roughly divided into strains with Usually present C
MV (CMV-P line etc.) is from Satellite 1 to RNA C
Even if this GMV, which belongs to the RNA4 component line that does not contain RNA5) (see A in Figure 1), is inoculated into plants such as tomatoes, no symptoms of rot will appear. However, CMV [C
MV-P (n) System 11 consists of a five-component system containing a component of molecule m (satellite RNA) (first
(See B in the figure). On the other hand, CMV [CMV-PF] exhibits only contagious symptoms on tomatoes, no erectile symptoms, and slight internal organs.
(f1) line etc.] also have five component RNAs and include satellite RNAs. ``In the present invention, an attenuated virus is created using satellite RNA isolated from the latter CMV strain that expresses only Kento symptoms and no paralytic symptoms. Such CMV strains include, for example, CM
V-PF (fl), CMV-P (m),
Examples include CMV-O (m).As a method for isolating satellite RNA, any conventionally known method for separating nucleic acid components from viruses may be applied.Generally, for example, viral proteins are denatured with appropriate reagents. After forcing
By repeating electrophoresis several times, satellite RNA
can be purified. Satellite RNA obtained from the above CMV strain has a molecular size of ~2.5X 105 Daltons, which corresponds to the component with the lowest molecular weight among various nucleic acid components in any strain (single-stranded or double-stranded). Main-stranded ribonuclei S
- Literature = Murant, A, F, and MaV
O, M, A, Ann, Rev, Phytopa
thol, 20:49-70.1982). In the present invention, this satellite RNA and satellite RN
Mix CMV with a four-component RNA component that does not contain A and inoculate vegetables, flowers, tobacco, and beans, and inoculate CMV of the RNA component.
A weakened virus with satellite RNA incorporated into the MV,
Produced and propagated within the plant body. The type of Satellite 1 to RNA and the four-component CMV strain used here can be arbitrarily selected, and various attenuated CMV viruses can be obtained depending on the purpose. Preferred combinations include CMV-PF (fl)-derived nosatellite RNA and CMV-P (No. 2>, CMV-P (No. 7>), CMV-P (pepper), CM
V-0 (No. 5), CMV-0 (No. 6), CMV-LH<N00
3), and the attenuated virus obtained by this combination can effectively control viral diseases caused by CMV infection in plants. On the other hand, for inoculation, attenuated viruses are created. Vegetables that can be used for propagation include flowers, tobacco, and legumes, such as tomatoes, tobacco, and green peppers. Eggplants, physalis, orientalis, cucumbers, melons, corns, soybeans, green beans, cowpeas, and zinnias. Chilling grass and pigweed are mentioned, but tobacco (variety = xanthus) has the highest growth rate of attenuated viruses.
It is preferable to use The method for producing weak ivirus is to use purified CMVl-g/
l, satellite RAS0.05~0.2 San+J/a+
Mix about 1% of phosphorus and add 0.1M phosphorus II buffer
) and then apply it to the surface of young tobacco leaves along with carborundum (400 to 600 mesh). The attenuated virus normally multiplies within the plant for about 7 days, after which the sap is collected from the plant. This sap contains enough C to inoculate plants as it is.
Since it contains enough attenuated virus to have a MV control effect, there is no need to particularly concentrate the sap or isolate the attenuated virus from the sap. Generally speaking, cowpeas are the source of attenuated viruses from their sap. The sap is inoculated onto the leaves of pigweed and trumpet, and after 2 to 3 days, the pits that appear are cut out, and the attenuated virus is removed by inoculating tobacco again and selecting plants that exhibit weak disease and mild fever. A typical nucleic acid component of such an attenuated virus is shown as C in FIG. The novel attenuated viruses obtained by the method of the present invention include the following. The name of the attenuated virus and its properties are listed below. CMV-P (No. 2) A produces extremely weak internal organs on tomatoes and spinach, mild mosaic appearance on green peppers and tobacco, and small groove spots on cowpeas. CMV-P (No. 7) A produces extremely weak internal organs on tomatoes and spinach, mild mosaic appearance on green peppers and tobacco, and large pit spots on cowpeas. CMV-P (pepper) A produces extremely weak internal organs on tomatoes, mild mosaic and pimples on spinach, green peppers, and tobacco, and large fringe spots on cowpeas. CMV-0 (No. 5 > A): Light mosaic appears on tomatoes, spinach, and green peppers, but mosaic appears on tobacco, and large green spots on cowpeas. CMV-0 (No. 6) A shows on tomatoes, , t:'-ma>
Light mosaic appears on spinach and tobacco, and the pits on Naspea are large. CMV-LH (No. 3) A is not infectious on tomatoes, peppers, and spinach, causes light mosaic on tobacco, and has small yellow spots on cowpea. Now, the CMV weakly tropic virus into which the satellite RNA has been incorporated can effectively control viral diseases caused by highly virulent CMV in vegetables, flowers, tobacco, and beans. As a control method, the sap collected from a plant that has been inoculated with the attenuated virus of the present invention and propagated may be directly inoculated to the target plant, or the sap collected from the plant inoculated with the attenuated virus of the present invention may be directly inoculated into the target plant, or the sap may be directly inoculated into the target plant. , 0) The attenuated virus diluted to 100 times FIO may be inoculated to the target plants along with a small amount of carborundum (400 to 600 mesh). In the inoculum containing the above-mentioned attenuated virus, the concentration of the attenuated virus is not particularly limited, but in order to reliably control the viral disease caused by CMV, it is preferable that the attenuated virus concentration is 100 times or more. Further, the site where the attenuated virus inoculum is inoculated onto a plant is not particularly limited, but in order to increase the inoculation efficiency, it is preferable to inoculate the surface of young leaves of the plant immediately after expansion. The plants to be inoculated with the attenuated virus may be at any growth stage, but preferably young seedlings are inoculated. Usually, about 10 to 15 days after vaccination, C.
Even inoculation with the virulent MV virus is effective and no severe internal symptoms appear. Furthermore, even if a plant inoculated with the highly virulent virus of the present invention is planted in a field and then inoculated with a weakly virulent virus, only a slight internal organ disease will appear. Moreover, vegetables and flowers that have been inoculated with attenuated viruses. Even when tobacco and beans are grown, there is almost no difference in yield compared to healthy plants that are not infected with any virus. [Effects of the Invention] According to the present invention, CMV that does not cause symptoms in tomatoes
An attenuated CMV virus can be easily produced by simply inoculating plants with the satellite RNA of the 14-component CMV that does not contain the satellite RNA. Moreover, by appropriately selecting the type of satellite RNA and the CMV strain, it is possible to easily obtain an effective, stable, and attenuated virus suitable for the purpose. Furthermore, the CMV control method using this attenuated virus is
Unlike methods such as spraying chemicals against vector insects of MV or preventing them from flying in, there is no risk of causing environmental pollution or increased production costs, and there is no technical national seal such as breeding CMV-resistant varieties, so it is extremely effective. It is an economical and practical method with high pest control effects.

[Embodiments of the invention]

) Low speed centrifugation...Hitachi refrigerated centrifuge 20PR
was used. :Ultracentrifugation: Hitachi's Ultracentrifugal Machine 65P or International Ultracentrifugal Separator B-60 was used. The above method is just one example of CMV purification; there are many other methods, and purification is also carried out by other methods. . Satellite RNA Isolation 1, 4 rma ~8-9 purified CMV lgl was added with 0.11 ml of 10% sodium lauryl sulfate and 50%
Add 0.25 ml of sucrose and treat in warm water at 40°C for 10 minutes to break down the virus coat protein and remove CMV RNA.
and released satellite RNA. 2. Isolation of satellite RNA from CMV RNA
This was carried out by electrophoresis using a 2% polyacrylamide gel. The composition and preparation of polyacrylamide gels were performed according to the method of Loenig [Biochcm, J.
113:131-138 (1969)]. Polyacrylamide gel is 13.501x 1
A vertical flat plate of 0 cm x O, 3 cm was made. The free CMV solution prepared in 3.1 was layered on top of the polyacrylamide gel at 50 mM/gel. Electrophoresis was performed for 4-5 hours. Other electrophoretic details followed the method of Loenig (described above). 4.1! After pneumophoresis, the gel plate was washed with 0.05% toluidine blue 0 (5511M sodium acetate, 0.1111M
containing EDTA) and decolorizing solution (5511).
After decolorizing with 1M sodium acetate, 0.11M EDTA), the satellite RNA band was cut out with a razor, placed on top of the polyacrylamide gel prepared in 2, and repeated twice in the same volume as 3. . 5. The finally excised satellite RNA band was ground using a homogenizer, and the satellite RNA was extracted from the polyacrylamide gel by a method using phenol and ethanol. Finally precipitated RN
A is TEN (0.11M sodium chloride, 20
mM Tris-HCl, 1 mM EDTA1PH8,
5) and stored at -70°C until use. Details of gel plate staining and satellite RNA extraction from the gel are described in the method of Peden and Symons [
Virolog 53: 487-429. (1
973) ] I went there. Development of CMV attenuated virus (see Figure 2) Laboratory tests on CMV attenuated virus are shown in Table 1.2 below. l1Il11i test against CMV attenuated virus Table 1 below. Il, III.

[Brief explanation of drawings]

FIG. 1 shows the results of electrophoresis of purified CMV using polyacrylamide gel to separate Eils nucleic acid. A: CMV-P strain: Nucleic acids of many CMV strains are R
Consists of four components NA1 to RNA4, satellite RN
There is no A. B: CMV-P (fl) strain: RNA1 to RNA
In addition to 4, it contains satellite RNA and is composed of 5 components of nucleic acids. C: Attenuated strain: Nucleic acid of satellite RNA incorporating satellite RNA of CMV-P strain and cMV-P (fl) strain. FIG. 2 is a diagram illustrating a method for producing one attenuated strain of CMV.

Claims (4)

[Claims] (1) Molecular weight 0.5 to 2.5 x 10^5 obtained from cucumber mosaic virus that does not show symptoms of macho on tomatoes
An attenuated cucumber mosaic virus characterized by incorporating Dalton's satellite RNA. (2) The attenuated virus according to claim 1, wherein the satellite RNA is derived from CMV-PF (f1) of cucumber mosaic virus. (3) Molecular weight 0.5 to 2.5 x 10 obtained from cucumber mosaic virus that does not contain satellite RNA and cucumber mosaic virus that does not cause erythema symptoms on tomatoes
^5 Dalton satellite RNA is inoculated into vegetables, flowers, tobacco, beans, and then the attenuated virus is obtained from the juice of the vegetables, flowers, tobacco, and beans. Creation method. (4) Molecular weight 0.5 to 2.5 x 10^5 obtained from cucumber mosaic virus that does not show symptoms of macho on tomatoes
A method for controlling cucumber mosaic virus, which comprises inoculating seedlings of target plants in advance with an attenuated cucumber mosaic virus that incorporates Dalton's satellite RNA.