JP2908594B2 - Plant virus inoculation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an improvement of a method for inoculating a plant virus.

[0002]

2. Description of the Related Art When plant viruses infect crops such as tomatoes and melons, yields are reduced and quality is reduced. In addition, when viruses infect vegetatively propagating plants such as grapes, the damage only occurs continuously. It also plays an undesirable role as a source of infection.

[0003] In general, plants infected with the virus hardly heal naturally, and there is no healing agent. Therefore, as a method of protecting these plants from the virus, use of resistant varieties, transmission of infectious plants, etc. Removal, application of insecticides to vector insects and use of repellent materials, and use of attenuated viruses that inoculate plants in advance with attenuated viruses and impart virus resistance to the plants are performed.

[0004] Among these methods of protecting plants from viruses, the method of controlling with attenuated virus has a high controlling effect, is economical, and has no fear of environmental pollution. Accordingly, in recent years, it has been receiving much attention.

[0005]

However, in this method, when a plant virus is inoculated into a plant, the plant virus cannot be infected simply by spraying a suspension of the plant virus on the surface of the plant, and the plant can be inoculated with a suitable amount. Doing processing such as scratching,
It is transmitted only after the virus has entered the plant.

Conventionally, when a plant virus is inoculated into a plant, the plant virus suspension is artificially rubbed with an abrasive or a friction agent or the like by rubbing the plant virus suspension with a cotton swab, finger, glass stick or the like. This method is carried out by inoculating the portion after rubbing, or by directly injecting a plant virus suspension, but such a method requires human labor and a long time, and has a disadvantage that it is not efficient.

A method of spraying a suspension of a plant virus in which an abrasive is suspended with a spray gun at a constant pressure to damage a test plant and simultaneously inoculate a plant virus (Japanese Patent Publication No. 52-1982) No. 12,795) is also known, but this method is quick and efficient, but has the disadvantage that it requires a large amount of expensive plant virus and is not economical.

The inventors of the present invention have conducted intensive studies in order to solve these drawbacks of the conventional method. As a result, when a roller is pressed and rotated with a plant virus and an abrasive interposed on the surface of the plant, The present inventors have found that the plant can be efficiently and economically injured and inoculated with the plant virus by very simple means, and the present invention has been completed based on this finding.

That is, according to the present invention, a roller is pressed and rotated by interposing a plant virus and an abrasive on the surface of a plant,
A method of inoculating a plant virus, comprising injuring the plant body and inoculating the plant virus.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

First, plant viruses used in the present invention include tobacco mosaic virus in cultivation of tomato, cucumber green spot mosaic virus in cultivation of cucumber, and citrus tristeza virus in cultivation of citrus (Literature: Mitsuaki Kametani, 1989). Agriculture and horticulture "64
Volume, pp. 159-164, and Hiroshi Tochihara, 1986
In addition to attenuated viruses such as "Agricultural Technology", vol. 41 (11), pp. 1-6), cucumber mosaic virus, tobacco necrosis virus, tobacco stem nematode virus, tobacco ring spot virus, tobacco dwarf virus, alfalfa mosaic virus , Tomato yellow spot virus, potato Y
Any plant virus, such as a virus, may be mentioned.

The plant virus can be inoculated at any time during the growth of the target plant, but in the case of controlling with an attenuated virus, the seedling of the plant is preferably used.

The method of inoculation is to spray a plant virus suspended in water or an appropriate buffer (for example, a 0.1M phosphate buffer near neutrality) by spraying it onto the leaves of the plant,
From above, a roller having an abrasive attached to the surface is pressed and rotated to injure the plant and inoculate the plant virus, or spray the abrasive on the surface of the target plant And spraying the plant virus suspension, and then contact and rotate the roller from above,
No.

As a roller obtained by adhering the abrasive to the surface, as shown in FIGS. 3 and 4, a cylindrical roller 1 is rotatably supported on a shaft 2, and the shaft 2 and a handle portion. 3 is integrally connected by a handle 4, and is obtained by applying a paste such as a water paste on the surface of the roller and uniformly adhering an abrasive on the paste.

Alternatively, in place of the above-mentioned "cylindrical roller 1", "a tape obtained by spirally winding an adhesive tape so that its surface becomes an adhesive surface to form a cylindrical shape" is used. An abrasive may be attached to the surface. This method
When the adhesive strength of the surface of the adhesive tape is reduced, the old surface is peeled off and the new adhesive surface is exposed, so that there is an advantage that the trouble of applying the glue to the surface of the roller can be omitted.

As the abrasive used in the present invention, any abrasive can be used as long as it can damage plants, especially leaves.
Celite or bentonite is particularly preferred. The particle size of these abrasives is preferably 50 microns or less, particularly 10 to 30 microns, because a high infection rate of a plant virus to a plant can be obtained.

In the following, in regard to this point, how the roughness of the abrasive attached to the surface of the roller affects the infection rate of the plant virus will be described with reference to experimental examples.

Experimental Example 1

Preparation of materials

Preparation of Plant Virus Tomato leaf 1 infected with attenuated cucumber mosaic virus 1
10 milligrams of the crushed liquid was inoculated into tomato cotyledons and the virus was propagated for about 1 to 4 weeks. After that, 150 grams of tomato-infected leaves were collected and frozen at an extremely low temperature of -70 ° C. After crushing 150 grams of the infected leaves, a 0.5 M citrate buffer (pH 6.0) containing 0.1% thioglycolic acid was used.
5) Add the same amount of chloroform as 300 ml,
Trituration was performed with a Waring blender to obtain a trituration solution containing virus particles.

This triturated liquid was centrifuged at 500 × g for 10 minutes, 120 ml of the upper layer (aqueous layer) was collected, and 12 g of 10% by weight powdered polyethylene glycol was added and dissolved therein. Particles were precipitated to facilitate precipitation.

This solution was centrifuged at 9,500 × g for 20 minutes, and the obtained precipitate (virus particles) was recovered.
A 0.05 M citrate buffer (pH 7.0) containing% Triton X-100 was added for washing and dissolved.

This was centrifuged at 12,000 × g, and the supernatant containing the virus particles was collected.
Xg was centrifuged for 45 minutes, and the obtained precipitate was suspended in 10 mM phosphate buffer to prepare a plant virus (attenuated virus of Cucumber mosaic virus).

Infected plant: cowpea (variety: black shark three shaku)

Abrasive: 150, 5 particles
Three types of silicon carbide corresponding to 0 and 25 microns (Carborundum # 100, # 280, and # 280 from Kishida Chemical Co., Ltd.)
600)

Inoculation method and results

When the leaves of cowpea (cultivar: black varieties three shaku) are inoculated with the attenuated virus of Cucumber mosaic virus, the attenuated virus is sprayed on the leaves of cowpea using the property of causing local spots on the inoculated leaves when infected. After spraying with, abrasive materials with different particle roughness are attached to the surface of the roller,
The roller was pressed and rotated on a cowpea leaf.

That is, cowpea was sown under the same culture conditions as described in Examples below, and about 10 days later, at the time when the primary leaves developed, 80 aliquots of the attenuated virus (concentration: 20 micrograms / milliliter) were used in each section. Of cowpea were sprayed in 10 ml portions.

Next, a roller (4.5 cm in diameter, 16 cm in length, see FIG. 3) having 4.5 g of water paste applied to the surface thereof is put on a roller.
0 g of abrasive was applied, and the roller was rotated from above the seedlings to injure the seedlings and to infect the attenuated virus.

Three days later, the number of spots appearing on the leaves was examined.

In the investigation of the number of spots, 10 cowpea were randomly selected from each section, and the number of spots appearing on the primary leaves of each strain was counted.

Table 1 shows the results.

[0033]

From these results, it is understood that the abrasive particles having a particle size of 50 μm or less are preferable because a high infection rate can be obtained.

Next, the concentration of the plant virus to be sprayed is 1
~ 100 micrograms / milliliter, preferably 2
0-50 micrograms / milliliter.

[0036]

According to the method of the present invention, a roller is pressed and rotated by interposing a plant virus and an abrasive on the surface of a plant body. The time required for inoculation is significantly reduced as compared to the stick inoculation method. In addition, a higher infection rate can be obtained with a smaller amount of inoculant than the method using a spray gun.

[0036]

The present invention will be described in more detail with reference to the following examples.

Example 1 In a nursery box (30 cm × 60 cm × 3 cm, see FIG. 2), a culture soil (bark: permucurite: peat moss = 3:
One liter of soil mixed at a ratio of 3: 2 was mixed with 7 g of uraform fertilizer (manufactured by Taiyo Bussan Co., Ltd.), and seeds of tomato (cultivar: K143) were sown in the greenhouse (25 ° C. in the daytime, 25 ° C. The seedlings were grown for 2 weeks at night (15 ° C.) to obtain tomato seedlings.

This was thinned out to about 200 per seedling box, and the attenuated virus of Cucumber mosaic virus obtained in the above experimental example was diluted with 50 mM phosphate buffer (p
H7.0, NH ₂ HPO ₄ , and NaH ₂ PO ₄ ), and the resulting suspension was sprayed (volume: 100 ml; see FIG. 1) to give a concentration of 25 micrograms / milliliter. 8 ml was sprayed and allowed to adhere to tomato leaves and stems.

Next, one roller was applied to a roller (4.5 cm in diameter, 16 cm in length, see FIG. 3) coated with 4.5 g of water paste.
0 g of abrasive (a mixture of celite: carborundum: bentonite = 1: 1: 1) was applied thereto, and the roller was rotated from above the seedling to injure the seedling and infect the attenuated virus. Was.

On the other hand, the same amount of the above-mentioned attenuated virus suspension was inoculated one by one by so-called artificial friction inoculation with a cotton swab instead of a roller.

After inoculation by both methods, each test plant was placed again in the greenhouse, and one week later, the presence or absence of infection was examined.

Table 2 shows the results.

[0043]

From the results shown in Table 2, it can be seen from the results of the conventional artificial inoculation using a cotton swab that the infection rate leaves only about 80% even if it takes 20 minutes, but according to the method of the present invention, it is 100% in 2 minutes. It can be seen that the infection rate can be obtained.

Example 2 In a nursery box (30 cm × 60 cm × 3 cm, see FIG. 2), culture soil (bark: permucurite: peat moss = 3:
One liter of soil mixed at a ratio of 3: 2 was mixed with 7 g of uraform fertilizer (manufactured by Taiyo Bussan Co., Ltd.), and seeds of tomato (cultivar: K143) were sown in the greenhouse (25 ° C. (15 ° C. at night) to give seedlings of tomatoes grown for 2 weeks.

This was thinned out to about 200 seedling boxes, and 10 g of an abrasive (a mixture of celite: carborundum: bentonite = 1: 1: 1) was uniformly sprayed on the thinned seedlings. (Sprinkle) and then,
The attenuated virus of the cucumber mosaic virus obtained in the above experimental example was added to a 50 mM phosphate buffer (pH 7.0, NH ₂
HPO ₄ , NaH ₂ PO ₄ ) to a concentration of 25 microgram / milliliter and a suspension (100 ml; see FIG. 1), and spray 8 ml of the suspension. Attached to tomato leaves and stems.

From above, a roller (4.5 cm in diameter)
, 16 cm long, see FIG. 3), while pressing the seedling, the roller was rotated to injure the seedling and inoculate it with the attenuated virus.

Next, the inoculated plants were placed again in the greenhouse, and one week later, the presence or absence of infection was examined. As a result, almost the same results as in Example 1 were obtained.

That is, the inoculation time was significantly shortened as compared with the conventional artificial friction inoculation method using a cotton swab, and a high infection rate of 96% was obtained with a small amount of inoculation solution.

[0050]

[Brief description of the drawings]

FIG. 1 is a schematic explanatory view of a spray (sprayer) used in the present invention, and FIG. 2 is a diagram of spraying a plant virus suspended in water or an appropriate buffer onto the leaf surface of the plant using the spray. FIG. 3 is a schematic illustration of a roller used in the present invention, and FIG. 4 is a schematic illustration of a roller used in the present invention. FIG. 3 is a schematic explanatory view showing a state in which a roller is rotated under pressure to injure the plant and inoculate the plant virus.

Claims (2)

(57) [Claims] 1. An inoculation of a plant virus, wherein a plant virus and an abrasive are interposed on the surface of a plant, a roller is pressed and rotated to damage the plant and inoculate the plant virus. Method. 2. The method of claim 1, wherein the abrasive has a particle size of 50 microns or less.