JP2000201535A - Inoculation of plant virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extremely simply scratch a plant body set on the ground surface with unevenness and inoculate the plant body with a plant virus so as to infect the plant body by 100% by scratching the surface of the plant body according to a specific method. SOLUTION: A brush 8 is applied to the surface of each plant body to scratch the surface thereof. Furthermore, the brush 8 is applied to the surface of the plant body to scratch the surface thereof and a plant virus liquid can be brought into contact with the surface before or after the scratching. The brush 8 having a polishing agent sticking thereto is applied to the surface of the plant body to scratch the surface thereof. The plant virus liquid can be brought into contact with the surface before or after the scratching.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a box (hereinafter referred to as "tray") in which tens to hundreds of small pots (hereinafter referred to as "cells") are connected in a grid pattern. In the tray raising method, in which the bed soil (cultivation soil) of the 9th minute) is placed, seeding is performed with a seeding machine, and a molded seedling is produced, rooted leaves are overlapped, and the seedlings (hereinafter referred to as “Rosette”) are flat and radially spread on the ground. Plant virus inoculated with a plant virus, which can very easily infect 100% of a plant virus in a plant seedling in a state.

[0002]

2. Description of the Related Art The present inventors sprayed a plant virus solution on the surface of a plant, and then pressed and rotated a roller to which an abrasive was adhered to damage the surface and to attach the plant to the plant. A method for inoculating the virus was developed, and a patent application was filed (JP-A-4-330005).

[0003] According to this method, the time required for inoculation is significantly reduced as compared with the inoculation method using a cotton swab, a finger, or a glass stick, and a smaller amount of plant virus solution (hereinafter referred to as "inoculation solution") is used as compared with the inoculation method using a spray gun. Has the advantage that a high infection rate can be obtained.

However, in this method, after spraying a plant virus solution on the surface of a plant in the process of raising seedling in the tray raising method, the handle 13, handle 12, and spinneret are applied to the surface as shown in FIG. A plant virus inoculation which comprises a handle 11 and a handle 13 connected to one end of a handle 12 and the other end of the handle 12 serves as a bearing for the roller 11; and the roller 11 is rotatably connected to the bearing. Even if the roller 11 of the vessel is pressed and rotated, a plant that grows with a large gap between the upper end portion of the seedling tray 4 and the floor soil surface of the cell 14 is rolled.
Since the outer peripheral surface of the la 11 could not be abutted, it could not be injured and had a disadvantage that the infection rate of the plant virus was low.

[0005] Seedlings having the property that root leaves overlap and spread radially flat on the ground (rosette state seedlings) generally require a relatively long number of days for raising seedlings. Each cell 1
4, the distance between the upper end of the seedling tray 4 and the surface of the floor soil 15 increases, and as the number of days for raising seedlings increases, the seedling 6 inside the cell hole 14 is formed by the cylindrical roller 11.
It becomes difficult to carry out the abrasion work of a. In addition, this method has a disadvantage that it is not easy to injure a plant planted on uneven ground.

[0006]

DISCLOSURE OF THE INVENTION The present invention is intended to extremely easily injure plants planted on uneven ground, especially rosette seedlings raised by the tray raising method, and to reduce plant virus by 100%. % Infectious,
It is intended to provide a method of inoculating a plant virus.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have finally completed the present invention.

That is, the present invention relates to (1) a method of inoculating a plant virus, which comprises contacting a brush with the surface of a plant to injure the surface. (2)
A method for inoculating a plant virus, which comprises bringing a brush into contact with the surface of a plant, damaging the surface, and contacting the surface with a plant virus solution before or after the brush,
(3) The present invention also provides a plant characterized in that a brush having an abrasive attached thereto is brought into contact with the surface of a plant to damage the surface, and a plant virus solution is brought into contact with the surface before or after that. This is the virus inoculation method.

Hereinafter, the present invention will be described in detail.

[0010] The plants to be used in the present invention include any plants, and crops capable of controlling the virus by inoculating attenuated virus, such as tomato, pepper, melon, sugar beet, pumpkin, tobacco, spinach, Vegetables such as lettuce, and flowers such as gentian, petunia, safflower, snapdragon, and pertussis are preferred.

As the brush used in the present invention, any brush can be used as long as it has a flexible or elastic wire material implanted on a base surface with a handle or handle. A brush for painting, a brush for toothpaste, a brush for clothes, a scrub brush, or a sword mountain using a fibrous metal wire which is rich in flexibility and elasticity. These are the size of each nursery bed,
It is preferable to use a plurality of them depending on the shape and area. When a plurality of brushes are connected and used, it is preferable that the required number of brushes are fixed on the same base surface in the vertical and / or horizontal direction, and the ends of the linear members are aligned.

The linear material used for the brush of the present invention preferably has an outer diameter of 0.05 to 0.25 mm and a length of 1 to 5 cm.

When the brush is brought into contact with the surface of the plant,
It is possible to damage the surface of the plant very easily. The contact may be merely applied to the surface of the plant, but a means for rubbing in the applied state is more preferable. Contact is
Although it is possible to perform the treatment once, it is preferable to perform the treatment 2 to 4 times in order to completely damage the surface of the plant.

Further, according to the present invention, when a brush having an abrasive attached thereto is brought into contact with the surface of a plant, a large amount of fine scratches can be formed on the surface of the plant, and the infection rate of virus inoculation can be increased. It is preferable because it is possible.

Examples of the abrasive include diatomaceous earth (celite), bentonite, and carborundum of 500 to 600 mesh. In particular, celite is uniformly dispersed in a liquid only by lightly stirring, and is electrically sprayed. The use of is preferred because the nozzle is not clogged. Their particle size is 5
~ 35 microns is preferred, and the amount added is 1-3
Add by weight.

In order to carry out the present invention, a brush is brought into contact with the surface of a plant to injure the surface, and before or after that, a plant virus solution is brought into contact with the surface.

The plant virus used in the present invention includes:
Infecting plants, especially crops, causes severe viral symptoms such as gangrene, mosaic, thread, dwarfing, yellowing, etc. and causes a significant decrease in yield. Citrus green spot mosaic virus, citrus tristezavirus in citrus cultivation (Literature: Mitsuaki Kametani, 1989 "Agriculture and Horticulture", Vol. 64, 159-1
64 pages, and Hiroshi Tochihara, 1986 "Agricultural Technology" 41
Vol. (11), pp. 1-6), soybean 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 virus, attenuated viruses of the above viruses, and the like.

Pre-inoculation of a plant (seedling) with the attenuated virus of the above plant virus has the advantage that damage to the above plant virus caused by the virus infection can be prevented.

The plant virus can be inoculated at any time during the growth of the target plant. However, if the purpose is to prevent the damage of the virus symptoms by using an attenuated virus, the plant virus must be inoculated in an external field. The seedling timing of the seedling is preferred, and the seedling raising time in a tray before the potting is particularly preferable because it can be inoculated intensively and efficiently. The optimal time for inoculation in the seedling raising tray is (1) cotyledon to two true leaves for tomato, (2) peppers to two true leaves for peppers, (3) melon and cucumber, one cotyledon to true leaves, ( 4) Petunia, eustoma, and tobacco crops, which have short plant heights and internodes and have seedlings in a rosette state, have four to six leaves, and (5) gentian have six to eight leaves.

The plant virus solution is preferably prepared by the following method. The squeezed solution of the infected leaves of the plant virus is filtered through a gauze, and the obtained solution is sterilized with water or an appropriate buffer (for example, a neutral buffer of about 0.1 M phosphate).
For example, the virus is prepared by diluting 5 to 50 times the weight of the infected leaf, or a purified virus solution (for example, cucumber mosaic virus solution) obtained by a conventional method is sterilized with water or an appropriate buffer to an appropriate concentration, for example. Virus concentration 25-5
Prepare by diluting to 00 μg / ml.

If necessary, a coloring agent is further added to the above-mentioned plant virus solution and suspended. The addition of the coloring agent has the advantage that it is easy to determine whether or not the virus solution has adhered after the plant virus solution has been brought into contact with the surface of the plant. As the coloring agent, any coloring agent such as a synthetic coloring agent and a natural coloring agent may be mentioned.In the case of an edible plant, it is preferable to use a food coloring agent in terms of safety, and the color is blue or red. Is preferable because the adhesion to the color (green) on the surface of the plant can be easily confirmed. The concentration (dilution concentration) of the colorant is, for example, 400 if a synthetic colorant is used.
Dilute ~ 800 times.

The means for contacting the plant virus liquid may be any method such as spraying, dipping, coating, and watering, but the most preferable is a means for spraying with a small amount of uniform spray and high inoculation effect. As a spraying means, a simple hand spray,
Examples include a power sprayer (for example, a small electric paint sprayer) and a spray gun. However, when using a simple hand spray, care must be taken because the spray port may be blocked by the abrasive. Small electric paint sprayer has discharge rate of 50-50
About 0 ml / min is preferable for performing a small amount of uniform spraying. Application rate of inoculum in the case of trays nursery (vertical 30, the case of the horizontal 60cm tray) is 5-15 mL, in the case of Chiyuka nursery, 3.3 m ² per 100~300ml are preferred.

Hereinafter, the present invention will be described more specifically with reference to the attached drawings. FIG. 1 shows a state in which a plant virus solution 5 is adhered to the surface of a plant (a seedling 6a in a rosette state) by a small electric painting sprayer 2, and FIG. 2 shows a state in which a brush 8 abuts on the surface of the plant. This shows a state in which the surface of a plant is injured. The seedling on the right side of the brush 8 shows a damaged state.

In FIG. 1, a small electric sprayer 2 containing a plant virus solution 5 (for example, Nippon Wagner Spray Co., Ltd.)
Using a mini painter manufactured by Tech Co., Ltd.), the plant virus liquid 5 containing a coloring agent and an abrasive added in advance is sprayed onto the surface of the seedling 6a in the rosette state of the seedling tray 4 and adhered evenly while observing the coloring condition.

Next, as shown in FIG.
a is rubbed several times in the vertical and horizontal directions with the brush 8 to which the abrasive has adhered.

FIG. 3 is a schematic explanatory view of the plant virus inoculation method of the present invention. The rosette seedlings 6a of the seedling tray 4 are brushed with the bristles 10 of the brush 8 at every corner of the foliage inside each cell hole 14. This indicates that the inoculation was rubbed all the way. Brush 8
Due to the elasticity, flexibility, and free movement of each hair 10, the foliage of the whole seedling, from each cell hole 14 to the foliage at the base of the seedling 6 a that does not stick to the surface of the seedling tray 4, The sprayed plant virus solution can be evenly rubbed and inoculated. In addition, the surface of the seedling tray 4 and the floor soil 15
Even when the distance from the surface (difference in unevenness) is large, it is easy to rub the seedlings 6 a inside the cell holes 14 with the bristles 10 of the brush 8. In FIG. 3, reference numeral 9 denotes a brush handle (or handle) which is used by hand when inoculating a remote seedling.

[0027]

The present invention will be described below in further detail with reference to examples.

Example 1 (1) Example of breeding seedlings of crops to be inoculated with attenuated virus First, as a culture soil for tomato, pepper, petunia and tobacco, "Cell Top Mix" manufactured by Honen and Sakata were used. "Super Mix A" manufactured by Seed Co., Ltd. was prepared by mixing 1: 1 and fine grain Kanuma soil, fine grain Akadama soil, and peat moss were mixed 1: 1: 1 as a culture soil for gentian. And two types of seedling trays (Yama-vegetable tray 25-200 (cell size 25 mm square × 45 mm depth), Yanma-vegetable tray 3 corresponding to the seedlings of each crop.
0-128 (cell size 30 mm square x 45 m depth
m) "and seeded with each crop seed. After germination, normal seedling management was carried out by injecting liquid fertilizer or watering under an appropriate temperature and lighting conditions according to the seedling of the crop, and each seedling was grown until a suitable growth period at which the attenuated virus was inoculated. The optimal time for inoculation is around the time when germination occurs and the seed grows and grows into a root pot to have several leaves, and it is appropriate 5 to 10 days before shipping as tray seedlings. The results of examining the growth of the seedlings at this inoculation time and examining whether the seedlings are compatible with the inoculation of the present invention are shown.

Table 1 Raising seedlings Raising seedlings ** Plant height Crops Tray days Number of plants Number of leaves Number of hills Above-ground area On tray surface (hole) (day) (stock) (sheets) (cm) (cm) Tomato 200 20 10 1.5 5.2 4.7 * (Momotaro Eight) P-Man 200 30 10 1.2 3.6 3.0 (Tosa wig) Petunia 128 40 21 6.7 2.0 1.2 (Titan Parade) Tobacco 200 40 10 5.7 1.5 0.9 (xanthin nc) Gentian 128 75 14 7.6 1.1 0.4 (Ezo Lindau) * () indicates the variety name, trade name, etc. of the seeded seed. ** The number of leaves indicates the number of leaves excluding tomato and pepper, and the number of leaves indicates the number of leaves one by one for gentian.

From the results shown in Table 1, the tomato and pepper seedlings protrude from the inside of the cell hole onto the surface of the seedling tray, and the cotyledons and true leaves are about 3 cm or more on the surface of the seedling tray. In the case of rubbing inoculation with a cylindrical roller described in JP-A-4-330005, the foliage of seedlings can be sufficiently rubbed on the surface of a seedling tray. However, petunia, tobacco and gentian, whose seedlings are in a rosette state, have a very low height of 0.4 to 1.2 cm on the tray surface, about half of the height is inside the cell hole, the internodes are extremely short, The number is large and the number of sheets is large, and it is not possible to inoculate the rollers by the conventional inoculation method by sufficiently rubbing the stems and leaves inside the cell holes.

(2) Comparison of inoculation results of attenuated virus in seedlings in a rosette state Purified solution of attenuated virus of cucumber mosaic virus ("NDM-1", manufactured by Nippon Del Monte Co., Ltd.) was diluted in phosphate buffer at a concentration of 100 μg / ml. Colorants (synthetic colorants,
(Brilliant blue-FCF) was added at 500 times to prepare an inoculum. In the present invention, the inoculum is sprayed on the seedlings by a simple hand spray on petunia of 6 to 7 seedlings grown on a 128-hole tray for 40 days, and a small amount is sprayed uniformly while observing how the foliage and leaves are colored blue. Immediately after spraying, brush with diatomaceous earth (celite) adhered to the tip of a brush (manufactured by Fuji Brush Co., Ltd., brush for paint, bristle length: 3.6 cm, brush width: 7 cm, outer diameter of brush bristles: approx. 1mm, the hair material is goat), and the brushes (brush width 30cm) created as shown in Fig. 3 are used to make a total of 3 foliage of the whole seedling up to the foliage inside the cell hole in the vertical and horizontal directions. Rubbed twice.

[0032]

COMPARATIVE EXAMPLE Comparative Example 1 Immediately after spraying the inoculant in the same manner as in Example 1 above, seedling trays were placed on a roller to which diatomaceous earth (Celite) was adhered (rolled by Nitoms, roller length 16 cm). The stems and leaves of the seedlings protruding on the surface of the seedling were rubbed a total of three times.

[0033]

Control Example 1 Control Example 1 The inoculant obtained in Example 1 was sprayed, and no rubbing operation was performed.

Control Example 2 Diatomaceous earth (Celite) was mixed with 2% of the inoculum obtained in Example 1 above, and a cotton swab (manufactured by Johnson & Johnson) was immersed in the liquid and placed on the back of the leaves of the seedlings. The leaf was rubbed with a cotton swab six times for each strain, and the cotton swab was replaced after each inoculation of eight strains. On the 8th day after the above inoculation operation, the strains to be examined were randomly taken from each of the 128-well trays, and attenuated viruses were detected.

The results of Example 1, Comparative Example, Control Example 1 and Control Example 2 are shown in Table 2.

Table 2 Investigation per tray Inoculation damage Detection of attenuated virus Rubbing device Inoculation solution Inoculation time Inoculation time Number of strains Number of strains detected * Infectious rate (ml) (min) (share) (share) (share) (%) Invention brush 8 0.75 13 0 13 100 Comparative Example Roller 8 1.08 13 4 11 85 Comparative Example 1 None 8 0.21 1000 0 Control Example 2 Swab 19 25.33 10 0 10 100 * * of attenuated virus On the 8th day after inoculation, 6 leaf pieces were taken from each strain, placed in a vinyl sachet, crushed, injected with 700 μl / bag of phosphate buffer, and mixed with celite. Immediately after the cotton swab was immersed in the solution, it was rubbed against the primary leaves of cowpea (variety: black shark 3 shaku), and the virus was detected when spots were generated, and was not detected when spots were not generated. The numbers indicate the virus detection results.

From the results shown in Table 2, the inoculation with the roller of the comparative example is as follows. First, the leaves of the seedling protruding from the seedling tray surface are pinched between the tip of the corner of the cell hole and the roller. 31% (out of 4 strains / 13 strains) to the extent that they were slightly torn. In addition, the rollers did not touch the foliage inside the cell hole of the seedling tray, the rubbing operation was not complete, and the infection rate was only 85%. In the inoculation of the control example 1 by spraying only the inoculant, no infection was possible because there was no operation of rubbing the seedlings. In Comparative Example 2, the inoculum was rubbed with a cotton swab evenly every six pieces per plant, so that a large amount of inoculum was required and the inoculation time was greatly required. On the other hand, according to the present invention, a small amount of spray liquid adhering to the seedling is applied to a 128-hole tray with a vertical width (about
m) The brush is simply rubbed several times with a brush, so that the inoculation time is short, and the free movement of the flexible hair tip of the brush against the unevenness of the seedling tray is sufficient to reach the foliage inside the cell hole. Seedlings have very little abrasion, no inoculation-damaged strains, and 1 attenuated virus
00% infection was achieved. Therefore, according to the present invention, a small amount of inoculant can be rubbed and inoculated quickly, intensively and efficiently by a simple instrument and a simple method, and the tray seedlings can be rubbed evenly and the plant virus can be completely removed. It can be infected and can be inoculated without damaging the seedlings due to the flexible movement of the brush tips.

(3) Comparison of Virus Propagation by Leaf Site of Attenuated Virus-Inoculated Strains Two days after the attenuated virus inoculation, two petunias of the present invention and the comparative example were sprinkled on the upper, middle and lower leaves, respectively. Separate the leaves into two small pouches, crush them, pour phosphate buffer solution, mix with celite, and immediately immerse a cotton swab in the solution. After inoculation by rubbing one by one, four days later, the number of spots generated on each primary leaf was counted, and the virus growth by site of the leaves of the attenuated virus inoculated strains of the present invention and the comparative example,
The number of cowpea spots per petunia strain was compared. Table 3
The results of the present invention and comparative examples are shown in FIG.

Table 3 Number of Cowpea Spots Occurred on Petunia Leaves Number of Investigation Strains Lower Leaf Middle Leaf Upper Leaf Total (strain) (q) (q) (q) (q) Present invention 10 19.0 22.8 20. 0 61.8 Comparative Example 10 1.6 13.7 16.1 31.4

As shown in Table 3, the number of spots of cowpea of the comparative example inoculated with a roller was small in the lower leaves, and increased in the number from the middle leaves to the upper leaves. This indicates that the roller could not rub the stems and leaves inside the cell holes in the recesses of the seedling tray, and that the attenuated virus did not easily proliferate in the lower leaves of the seedlings. On the other hand, in the present invention inoculated with a brush, the foliage inside the cell hole is sufficiently rubbed by the free movement of the flexible hair tip of the brush with respect to the unevenness of the seedling tray, and the lower leaves, Approximately equivalent spots were generated from both the middle leaf and the upper leaf, and the total number of cowpea spots per petunia strain was double that of the comparative example, indicating that the attenuated virus quickly penetrated into the petunia body and proliferated. .

[0041]

According to the present invention, a small amount of a plant virus solution is provided.
Using a simple device such as a simple spraying device or brush, spray the seedlings evenly and then rub the seedlings several times.
This has the effect that the inoculation of a large amount of seedlings can be performed quickly, efficiently and intensively in a short time. The present invention also relates to tomato,
In addition to crops that have leaves protruding completely from the seedling tray surface, such as man, the internodes of petunia, eustoma, tobacco, gentian, etc. are extremely short, and the leaves overlap, forming a rosette state, and the leaves are completely on the surface of the seedling tray. Even for seedlings that have not emerged, rub the brush from the leaves at the tip of the seedlings on the surface of the seedling tray to the foliage at the groundside inside the cell hole by the free movement of the brush with the flexible fine tips. Has the effect of being able to inoculate. In addition, the seedlings can be inoculated without damaging the plants, and furthermore, the plant virus can quickly penetrate into the plant body and almost completely infect. The present invention is not only in the case of inoculating a plant virus on a plant planted on flat ground,
A plant virus can be inoculated and infected without uneven inoculation of a plant planted on uneven ground.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram schematically showing a spray state of a plant virus solution of the present invention.

FIG. 2 is a conceptual diagram schematically showing inoculation by the brush of the present invention.

FIG. 3 is a cross-sectional view of a rosette-shaped seedling in a seedling tray to which a plant virus solution has adhered, being rubbed with the brush of the present invention and inoculated.

FIG. 4 shows a conventional method of inoculating rosette-shaped seedlings in a seedling tray to which a plant virus solution has adhered (JP-A-4-330005).
FIG. 4 is a cross-sectional view of a situation in which the inoculation is performed by rubbing with a roller.

[Explanation of symbols]

 2 ... Electric sprayer 4 ... Seedling tray 5 ... Plant virus solution 6a ... Seedling in rosette state 8 ... Brush 9 ... Brush handle 10 ... Brush hair 11 ... Cylindrical roller 12 ... Roller handle 13 ... Roller La handle 14 ... cell 15 ... floor soil

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Eiji Ishimura 4-13, Koamicho, Nihonbashi, Chuo-ku, Tokyo F-term in Delmont Corporation (reference) 2B022 AA01 AB11 AB15 AB17 DA19 2B030 AA02 AB03 AD05 CG05 4H011 AA01 AA04 DA13 DD03 DE17

Claims (6)

[Claims] 1. A method of inoculating a plant virus, wherein a brush is brought into contact with the surface of a plant to damage the surface. 2. A method for inoculating a plant virus, comprising: bringing a brush into contact with the surface of a plant to damage the surface, and contacting the surface with a plant virus solution before or after the brush. 3. An inoculation of a plant virus, wherein a brush having an abrasive attached thereto is brought into contact with the surface of the plant to damage the surface, and a plant virus solution is brought into contact with the surface before or after the brush. Method. 4. A brush in which a large number of linear materials having an outer diameter of 0.05 to 0.25 mm are implanted on a base surface.
3. The method for inoculating a plant virus according to any one of 3 above. 5. The method according to claim 1, wherein the plant is a rosette vegetable seedling, a flower seedling, or a fruit seedling. 6. The method for inoculating a plant virus according to claim 1, wherein a plant virus solution to which a coloring agent is added is used as the plant virus solution.