JP3313846B2 - Plant disease protective agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for protecting plant diseases, and more particularly to an agent for protecting plant diseases caused by Pseudomonas syringae.

[0002]

BACKGROUND OF THE INVENTION Syringomycin, a plant toxin produced by Pseudomonas syringae , is an adzuki bean, cowpea,
It causes characteristic symptoms in many crops such as onions and peaches, and significantly reduces their productivity and commercial value. Conventionally, pesticides such as fungicides have been sprayed to control the disease caused by Pseudomonas syringae, but there are many problems regarding its safety. In addition, the control effect is not sufficient, and it is difficult to control the disease, especially for crops with limited application of pesticides, and it is even more difficult to treat those crops once infected. Was.

[0003]

Accordingly, the present inventors have developed a substance which has a protective effect against the cytotoxicity of syringomycin, a phytotoxin produced by Pseudomonas syringae, and can be expected to prevent and treat diseases. As a result of intensive studies, the present inventors have found that sterols that are safe for animals and plants are extremely effective for the above purpose, and have completed the present invention.

[0004] That is, the present invention provides an agent for protecting plant diseases caused by Pseudomonas syringae, which contains cholesterol as a main component.

[0005] The plant of the present invention comprising cholesterol as a main component
When using an object disease protectant into a plant, which is the main component co
The sterol may be mixed with a suitable carrier and then sprayed or applied to plants in the form of emulsions, powders, granules, wettable powders or the like, or absorbed directly from the roots. Further, if desired, a surfactant or the like can be added to improve the permeability and spreadability to plants.

The concentration of cholesterol in the above protective agent is 10 to 10,000 ppm, preferably 10 to 10 ppm.
１，1,000 ppm. This concentration can be appropriately adjusted depending on the type and situation of the plant. The protective agent is preferably used in accordance with the time of the onset of the disease, but may be used prior to the onset of the disease for the purpose of preventing the disease. In addition, for orchards in which it is not easy to remove the diseased site, the above-mentioned protective agent can be used for the purpose of treatment throughout the year.

[0007] The crops to which the protective agent of the present invention is applied are not limited. For example, rice,
Cereals, wheat, legumes, tea, tobacco, leafy vegetables, fruity vegetables,
Root vegetables, fruit trees, flowering plants, and the like.

[0008]

The present invention will be described below in detail using plant tissue culture cells. Example 1 1) Production and Purification of Syringomycin Pseudomonas syringae grown in a Pseudomonas syringe storage medium (see Table 1) was inoculated into 5 mL of a syringomycin production medium (see Table 2) at 25 ° C. For 1 day to obtain a preculture. For the main culture, 1.5 L of medium
The mixture was dispensed into 10 00 mL Sakaguchi flasks, and 5 mL of the preculture solution was added per flask, followed by stationary culture at 25 ° C. for 4 days.

[0009]

[Table 1]

[0010]

[Table 2]

After the culture, the cells are collected by centrifugation (6000 rpm × 10
Min), first, syringomycin was extracted from the cells. The cells were suspended in 50% (v / v) acidic acetone (adjusted to pH 2.0 with 1N HCl) to extract syringomycin. This solution was centrifuged (6000 rpm × 10 minutes) to remove cells, and the supernatant was concentrated under reduced pressure to remove acetone. To this was added n-butanol, and the butanol layer was separated. This was again concentrated under reduced pressure. Next, syringomycin was extracted from the supernatant,
After adjusting the pH to 2.0 with 1N HCl, n-butanol was added, and the emulsion between the butanol layer and the aqueous layer was centrifuged to collect the butanol layer. This was concentrated under reduced pressure. Thereafter, the total amount of the liquid extracted from each of the cells and the supernatant was adjusted to 0.
The volume was made up to 300 mL with 1% trifluoroacetic acid (TFA). After adsorbing this through Amberlite XAD-2,
0.1% TFA: linear concentration gradient of 2-propanol (0 to 1
00%) and fractionated. The activity of each fraction was investigated by the following bioassay, and the active fractions were collected and freeze-dried.

2) Bioassay method 8 × 10 6 strains of 8A-1B were added to 2 mL of soft agar and spread on 20 mL of YEPD agar medium. To this, six holes were formed in a regular hexagonal shape with a cork borer (10 mm), and a 50 μL sample was injected into each hole, and the activity was determined from the size of the inhibition circle.

3) Syringomycin protection test using cholesterol A test was carried out using cultured cells of tobacco (Nicotiana tabacum cv. Xanthi nc). The cultured cells used for the test were B5
Naphthalene acetic acid (NAA) in medium (Gamborg, 1966)
mg / L, benzyl adenine (BA) 0.02 mg /
L, subcultured every 7 days in a liquid medium containing 3% sucrose.

On the third day after the passage, 2 μg / mL of syringomycin and 4 μg / mL or 40 μg / mL of cholesterol were added, and the cells were cultured for 3 days or 6 days. Cell inoculation concentration at the start of administration is Packed Cell
The volume (compressed cell volume) was 0.3 mL / dL. After the treatment, the TTC reducing ability was measured. It indicates the strength of dehydrogenase in cells and is a measurement method used as an indicator of respiratory activity.

The measuring method will be specifically described below. Remove the medium from the cell suspension and add 0.8% TTC
Add 2 mL of phosphate buffer (pH 7.5) containing (2,3,5-triphenyl tetrazolium chloride) (the solution is degassed in advance). Reaction at 35 ° C for 2 hours, 2N
The reaction is stopped by adding 2 mL of sulfuric acid. Thereafter, the cells are removed, and TPF (1,3,5-triphenyl formazan) generated in the cells is extracted using 80% ethanol. 485 nm after extracting solution to 20 mL
Was measured to determine the activity (the amount of TPF produced per unit time). Tables 3 and 4 show the measurement results. When syringomycin was administered at 2 μg / mL, the respiratory activity of the cultured tobacco cells was only 1 to 3% of the control group, showing almost no activity. % Activity was maintained. The administration of cholesterol alone did not affect the respiratory activity, and the administration of 40 μg / mL resulted in a higher respiratory activity than the control group.
From these results, it was revealed that cholesterol has an effect of protecting cells from the cytotoxicity of syringomycin and is harmless to cells.

[0016]

[Table 3]

[0017]

[Table 4]

[0018]

EFFECT OF THE INVENTION Cholesterol has the effect of protecting plant cells from the cytotoxicity of syringomycin produced by Pseudomonas syringae. Therefore, according to the present invention, an effective and safe plant disease protective agent against Pseudomonas syringae can be established.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Tokichi Miyagawa 2-360 Kagamiyama, Higashihiroshima-shi, Hiroshima Hiroko-jukusha 1-304 (56) References JP-A-61-251601 (JP, A) JP-A-58 JP-A-51820 (JP, A) JP-A-5-194191 (JP, A) JP-A-63-126820 (JP, A) JP-A-64-40445 (JP, A) JP-A-1-191681 (JP, A) ) Bidwai, A .; P. , Et. a l. , Mechanism of action of Pseudomonas syringae Phyto xin, syringomycin. Stimulation of red beet pasm, Plant-Physiol. , (1987), Vol. 83, No. 1, pp. 39-43 Julmanop, Chanchai, et. al. , Protection by sterols against the cytotoxicity of syringomycin in the year Saccharomyces cerevisiae, J. et al. Gen. Microbio l. Vol. 139, No. 16, No. 16, 1993, UK. 10, pp. 2323-2327 (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 31/06 A01N 45/00 CA (STN) REGISTRY (STN) JICST file (JOIS)

Claims (1)

(57) [Claims] [1] An agent for protecting plant diseases caused by Pseudomonas syringae, comprising cholesterol as a main component.