JP2846610B2 - Screening method for elicitor that induces phytoalexin production in rice and rice disease controlling agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for screening an elicitor having the property of inducing the production of phytoalexin in rice, and a rice disease controlling agent comprising the elicitor screened by the screening method as an active ingredient. It is. More specifically, the present invention provides a method for rapidly and accurately screening elicitors that induce phytoalexin production in rice,
A test rice is cultivated, the test sample is applied to an appropriate part of the rice plant, and the specific phytoalexin produced in the rice plant is analyzed as an indicator for screening, whereby the rice is analyzed. And a method for screening a substance having a property of inducing the production of phytoalexins, such as phytocasan and momilactone. These phytoalexins have strong antibacterial properties against rice diseases, such as rice blast fungus and rice sheath blight fungus, so elicitors having the property of inducing the production of phytoalexins in rice are effective as rice disease controlling agents. Useful as an ingredient.

Further, the present invention relates to a cerebroside compound P having an action of inducing the production of phytoalexin in rice.
O8 and PO9, the cerebroside compound PO8,
A method using PO9 as a rice disease controlling agent, and further comprising culturing rice blast fungus in a liquid medium, separating the cells, extracting the cells with an organic solvent, and purifying the cerebroside compound by HPLC or the like. The present invention relates to a method for producing PO8 and PO9. The present invention also relates to a method for inducing the production of phytoalexins phytocasan and momilactone by applying the thus obtained cerebroside compounds PO8 and PO9 to the leaves of rice. Since these phytoalexins have strong antibacterial properties against rice diseases, for example, rice blast fungus and rice sheath blight fungus, the cerebroside compounds PO8 and PO9 are useful as active ingredients of rice disease controlling agents.

[0003]

2. Description of the Related Art In general, it is known that plants show a resistance reaction (hypersensitivity reaction) when they come in contact with a pathogenic bacterium, and produce phytoalexins, which exhibit antibacterial activity against the pathogenic bacterium, in tissues around the reaction site. . Phytoalexins of rice include momilactone A, B, oryzalexin A, B, C,
D, E, F, S, Sakuranetin, Orizalic Acid A, B, Orizalide A, B are known, and in addition, phytocasans A, B, C,
D (Japanese Patent Application No. 7-43520).

[0004] Substances that produce and induce phytoalexins in plants are called elicitors (Keen, NT; Scienc
e 187: 74-75 (1975)), many substances have been isolated from plant pathogens. A typical elicitor is Phytophthora megasperma f. Sp.g as a polysaccharide substance.
hepta-β-D-glucopyranoside isolated from lycinea
(Sharp, JK, B. Valentand, P. Albershim; J. Bio
l. Chem. 259: 11321-11336 (1984)).
Monicoline A isolated from onilinia fructicola (Cruic
kshank, IAM and DR Perrin; Life Sci. 7: 449
-458 (1968)), eicosapentaenoic acid isolated from Phytophthora infestans as a lipid (Bostock, RM, J.
Kuc and RA Laine; Science 212: 67-69 (1975).

[0005] As described above, elicitor has an action of inducing the production of phytoalexin, which has antibacterial activity against diseased bacteria in plants, and therefore has a high safety in controlling plant diseases by an action different from conventional pesticides. As a method for finding useful elicitors, which can be considered as a substance that can be an active ingredient of an agent, and for finding useful elicitors, a new search method capable of quickly and easily searching for a substance having elicitor activity is strong. Was sought.

[0006]

Under these circumstances, the present inventors have developed a novel elicitor for rice plants that seeks to induce the production of phytoalexins. Despite extensive research with the aim of developing a search method, it is extremely difficult to screen for substances with elicitor activity using individual rice plants, and no effective method has been established so far. I can say Thus, the present inventors have conducted various studies to develop an effective method, and have studied basic techniques such as the type of test rice plant, the method of growing rice, the method of applying test samples, and the method of analyzing phytoalexin. Has been successfully established, and it has been found that it becomes possible to screen for a substance having elicitor activity.

That is, as a result of examining the rice used for the test, the varieties and types of the test rice plants, the cultivation temperature / humidity,
It was found that the age of the test rice plant, the application site of the test sample, and the like were extremely important, and it was found that the screening method could be used by setting optimal conditions for each. For the phytoalexin to be analyzed,
Method for extracting phytoalexin induced in rice body and HPL using phytocasan and momilactone as preferred examples
The analysis method by C was determined. As a result of testing various substances by the above method, several compounds having elicitor activity were found.

Further, the present inventors have screened a wide variety of substances having elicitor activity for microbial products using the above-mentioned screening method. As a result, the product of rice blast fungus has such elicitor activity. I found that. Further, this substance was isolated by means of solvent extraction, column chromatography, etc., and two types of active substances were obtained, and as a result of conducting a structural analysis, the cerebroside compounds PO8 and PO9 having the following structural formulas were obtained. The present invention has been completed by clarifying that these substances are effective as an active ingredient of a rice blast control agent.

[0009]

Embedded image

The chemical structure of cerebroside compounds PO8 and PO9 discovered by the present inventors as substances having elicitor activity has already been reported, and PO8 is cerebroside A (Sitrin, RD et al .; J. Antibiot. 41; 4
69-480 (1988)) and PO9 is PENII (Kawai,
G. et al .; Agric. Biol. Chem. 49: 2137-2146 (198
5)), Cerebroside C (Sitrin, RD et al .; J. A.
ntibiot. 41: 469-480 (1988)). These reported substances are microbial products,
The microorganism is different from the rice blast fungus which is a bacterium producing PO8 and PO9.
There is no description of the elicitor activity of No. 9. Furthermore, the present inventors were the first to isolate these cerebroside compounds from the rice blast fungus, and there are no other reports.

The present invention relates to a method for screening an elicitor that induces the production of phytoalexin in rice, as described above, and using a rice seedling as a test plant, the phytoalexin produced in the rice body. The present invention relates to a method for analyzing HPLC (phytocasan, momilactone, etc.) by HPLC. The present invention also relates to an elicitor active substance obtained by this method. That is, an object of the present invention is to provide a method for screening elicitors that induce phytoalexin production in rice. Further, the present invention provides a screening method for quickly and easily searching for an elicitor having a property of inducing the production of phytoalexin in rice, using phytocasan of rice phytoalexin and momilactone A as indicator substances. It is intended for. Another object of the present invention is to provide a rice disease controlling agent comprising a specific substance having an action of inducing the production of phytoalexin in rice as an active ingredient.

Further, the present invention produces cerebroside compounds PO8 and PO9 by collecting cerebroside compounds PO8 and PO9 having the property of inducing the production of phytoalexin in rice from cultured cells of rice blast fungus. It is intended to provide a method. Another object of the present invention is to provide a rice disease controlling agent for controlling rice blast disease and rice crust blight disease by applying cerebroside compounds PO8 and PO9 to rice.

[0013]

Means for Solving the Problems The present invention for solving the above-mentioned problems is a method for screening elicitors for inducing the production of phytoalexins in rice, comprising the steps of using rice seedlings as test plants; Applying the sample to an appropriate site of the rice seedling, and screening the elicitor using the specific phytoalexin produced in the plant as an indicator substance, wherein the elicitor screening method, is there. In addition, the present invention also provides a test sample, which is applied drop-wise to the tip of the rice leaf of a young rice plant, and the phytoalexin produced in the plant is subjected to solvent extraction, and phytocasan of rice phytoalexin, momilactone A
The above-mentioned method for screening for elicitor is characterized in that the above-mentioned method is characterized in that analysis is performed by high performance liquid chromatography (HPLC) using as an indicator substance. The present invention also provides 2-pyrazinecarboxylic acid, picolinic acid, 2,6-pyridinedicarboxylic acid, and 7,3-pyridinedicarboxylic acid having an action of inducing the production of phytoalexins in rice screened by the above screening method. The present invention relates to a rice disease controlling agent comprising, as an active ingredient, one or more substances selected from an acid, pyrrole-2-carboxylic acid, oxonic acid, and cerebroside compound P08 or P09. Furthermore, the present invention provides a cerebroside compound PO8 or P8 having an action of inducing the production of phytoalexin in rice, which comprises culturing rice blast fungus and extracting the fungus with an organic solvent.
O9 manufacturing method.

[0014]

Next, the present invention will be described in more detail. The present inventors have examined in detail the varieties and types of test rice plants, cultivation conditions, phytoalexin analysis conditions, and the like as screening methods for substances that produce and induce phytoalexins in rice. Akitakomachi, Koshihikari, etc. are suitable as rice varieties to be used for the test, and cultivation is a granular cultivation appropriately containing nitrogen, phosphoric acid, and potassium necessary for growing rice, specifically, Hornens cultivation 1
It was found that control of temperature, humidity, and illuminance was important as conditions for cultivation of test rice plants, which were appropriate to use No. (all agriculture). Specifically, for example, seeds that have buds are planted in pots, cultivated in a dark room at 30 to 32 ° C. for about 2 to 3 days, and transferred to the artificial weather chamber when the buds extend about 2 to 3 cm on the soil. . The climate chamber has a temperature of 18-20 ° C and a humidity of 8
0-85%, illuminance 2000-3000lux. Next, when the third leaf comes out, the illuminance is 3000 to 4000.
lux, 95-100% humidity, daytime temperature 27-30 ° C, and cultivate until the sixth leaf is completely developed. It goes without saying that these cultivation conditions can be appropriately changed within the same range. In addition, a method in which the test sample is applied dropwise to the tip of the rice leaf of the sixth leaf age using a capillary pipette was determined as a suitable method.

After the rice to which the sample has been applied is further cultivated for one week, the leaves to which the sample is applied are shredded and extracted with a solvent such as ethyl acetate or methanol. The extract is analyzed by high performance liquid chromatography (HPLC), for example, phytocasan,
Regarding momilactone, phytocasan A having a retention time of about 35 minutes, phytocasan B having a retention time of about 43 minutes,
The amount of phytoalexin derived from the peak height of momilactone A at a retention time of about 50 minutes can be determined. Other phytoalexins can be analyzed in the same manner.

The above screening method basically has the following configuration. (1) Test plant (rice) Varieties: Akitakomachi, Koshihikari Age: Young plant, especially the 6th leaf Cultivation: Cultivation until the 6th leaf is fully developed

(2) Application of test sample Application site: rice leaf, especially its tip Application method: dripping with a capillary pipette etc. Cultivation period after application: about 1 week

(3) Extraction and analysis Sample preparation: Shred the leaves of the application site Extraction: Solvent extraction with ethyl acetate, methanol, etc. Analysis: High performance liquid chromatography (HPLC) analysis

According to this screening method, a compound as shown in Example 2 to be described later was found as a substance capable of producing and inducing phytoalexin in rice.

Furthermore, the present inventors applied a sample to the leaf surface of rice, cultivated it in a timely manner, and aimed at searching for a substance that produces and induces phytoalexin using the above screening method. In the process of performing various tests to measure the amount of phytoalexin produced in the body,
It was found that the organic solvent extract of the rice blast fungus showed high activity to induce phytoalexin. Therefore, the active ingredients (PO8, PO9) are obtained by extracting the cells of rice blast fungus with a solvent such as ethyl acetate, acetone, ethanol or the like, and purifying them by means of high performance liquid chromatography, thin layer chromatography or the like. By processing, it can be separated as a single component.

As the liquid culture for culturing the rice blast fungus used in the present invention, any medium which uses an extract of a plant or a microorganism and which has been conventionally used for culturing filamentous fungi can be used. , Preferably, for example, P
An SY medium and the like are exemplified. Plant extracts such as potatoes, yeast extracts and the like are used in these media.

In order to obtain the cells of the rice blast fungus containing the active ingredient, the blast fungus may be inoculated into an appropriate liquid medium as described above and cultivated at 28 ° C. and 180 rpm for 7 days, for example. .

As a specific process for extracting and separating PO8 and PO9, for example, after extracting the cells of rice blast fungus with a solvent such as ethyl acetate, TSKgel ODS120A,
A method of purifying and isolating by a purification process such as fractionation by high performance liquid chromatography (HPLC) using a column such as ODS120T (manufactured by Tosoh Corporation) and concentration to dryness is exemplified as a suitable method, but is not limited thereto. In addition, it is also possible to carry out by appropriately combining other similar purification means, and the purification process is not particularly limited.

The PO8 and PO9 according to the present invention have the following properties. 1) According to mass spectrometry by FAB-MS, PO8 has a molecular weight of 7
25 and PO9 had a molecular weight of 753. 2) PO8 and PO9 each indicate the infrared absorption spectrum shown in FIGS. 3) PO8 and PO9 are each ¹ H shown in FIGS.
1 shows an NMR spectrum. 4) PO8 and PO9 are each ¹³ C shown in FIGS.
1 shows an NMR spectrum. 5) PO8 and PO9 have an effect of inducing phytoalexin production in rice. The phytoalexins to be derived include phytocasans A, B, C, D, momilactone A, B and the like. Since these phytoalexins have strong antibacterial activity against rice blast fungus and rice sheath blight fungus, rice plants induced with these substances are considered to exhibit resistance to disease germs.

Since PO8 and PO9 according to the present invention have the property of inducing the production of the antibacterial substance phytoalexin in rice, as shown in the examples described later,
8, PO9 is useful as an active ingredient of a rice blast disease controlling agent and a rice wilt disease controlling agent. That is, by applying the compound to rice as a drug in an appropriate form, it is possible to prevent the occurrence of rice blast and rice sheath blight.

The drug of the present invention may be formulated into an appropriate form containing the above-mentioned effective amount according to the purpose of use, as shown in the examples described later. There is no particular limitation. As a method for applying a compound having an action of inducing the formation of phytoalexin to rice, as described in Examples described later, for example, the compound may be prepared by adding 0.1% of Tween 20 at pH 7.0.
0 is dissolved in 20 mM phosphate buffer, and the solution is sprayed and sprayed on rice.
The present invention is not limited to this, and other methods may be used. The form of a drug for applying the compound to rice, the form of use, the method of application, and the like are not particularly limited.

[0027]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. Example 1 (1) Cultivation of rice used as a test plant Seeds of rice (variety: Koshihikari or Akitakomachi)
Was filtered out with salt water to remove bad seeds, and then subjected to a sprouting treatment. The seeds that have budded out are honens cultivated soil No. 1 (all agricultural).
Eight grains are planted in the No. 6 pot where water was soaked from below,
Cultivated in a dark room at 32 ° C. for about 2-3 days. 2 buds on the soil
When it was extended to about 3 cm, it was transferred into a glass case in a climate chamber. The climate chamber was kept at a temperature of 18 ° C., a humidity of 80%, and an illuminance of 3000 lux, but the rice pot was placed in a slightly light-shielded place. Next, when the third leaf comes out, the illuminance 3
The conditions were changed to 000 lux, 100% humidity, daytime temperature of 27 to 30 ° C, and cultivated until the sixth leaf was completely developed. The rice seedlings thus cultivated were subjected to the following tests.

(2) Application of Sample and Induction of Phytoalexin Samples 1 to 7 were prepared as shown in Table 1. Using a capillary pipette, 20 μl of the sample solution was applied to 10 points (site) at the tip of the sixth leaf of the rice seedling cultivated as described above. Sample is 0.1
% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) at pH 5.5
(Sample 1) or 20m at pH 6.5 (Samples 2 to 7)
A predetermined concentration was dissolved in an M phosphate buffer and used. The rice to which the sample was applied had a humidity of 80%, an illuminance of 2000 lux and a night temperature of 18
C., at a daytime temperature of 25 to 26.degree. C. for 3 days. Furthermore, cultivation was continued for four days with the humidity set to 100% during the day.

(3) Extraction and Analysis of Phytoalexin Eight rice leaves to which the sample was applied were cut, cut into pieces, added with 5 ml of ethyl acetate and 5 ml of 0.1N Na ₂ CO ₃ and subjected to shaking extraction overnight. . The extract was processed by a centrifuge (5000 rpm).
m, 4 ° C, 20 minutes), and the ethyl acetate phase was separated and concentrated to dryness. The residue was dissolved in 0.4 ml of ethanol.
6 ml of 0.02N HCl was added and mixed. This was centrifuged, and 100 μl of the supernatant obtained was subjected to HPLC analysis. HPLC conditions are as follows. Column: TSK-gel ODS 120T (4.6 mm × 300 mm) Solvent: Acetonitrile (45): water (55) (volume ratio) Flow rate: 1.2 ml / min Temperature: 50 ° C. Detector: UV 280 nm (phytokasan) / 215 nm (momilactone)

(4) Results Under these conditions, the phytoalexins induced in rice leaves by Samples 1 to 7 were as shown in the table below.

[0031]

[Table 1]

As shown in Table 1, Samples 1 to 7 contain a substance having an action of inducing the production of phytoalexin in rice.

Example 2 (1) Identification of a substance having an action of inducing the formation of phytoalexin in rice As a result of analyzing the active ingredients of the above-mentioned samples 1 to 7, the following compounds were found in phytoalexin in rice. It has been identified as a substance that has the effect of inducing the production of proteins. Phytocasan EL (shown by the following structural formula) 2-Pyrazinecarboxylic acid (2-Pyrazinec)
arboxylic acid Picolinic acid 2,6-pyridinedicarboxylic acid (2,6-Pyri)
dicedicarboxylic acid) 7,3-pyridinedicarboxylic acid (7,3-pyri)
dicedicarboxylic acid pyrrole-2-carboxylic acid (Pyrole-2-c)
aroxylic acid Oxonic acid potas
sium salt)

[0034]

Embedded image

Of the above compounds, phytocasan EL
Is a new natural substance isolated from rice and has the following properties. 1) It is a colorless gum, soluble in acetone, chloroform, methanol and ethanol, and 100
Dissolves at concentrations around ppm. 2) Precise molecular weight by high resolution mass spectrometry is 316.20
62 (calculated as C ₂₀ H ₂₈ O ₃ 31.6205)
showed that. 3) An infrared absorption spectrum shown in FIG. 7 is shown. 4) It has the effect of inducing phytoalexin production in rice. 5) It has an effect of inhibiting germination of spores and elongation of hyphae of rice blast fungus. The concentration of phytocasan EL, which inhibits spore germination by 50%, is 7 ppm, and at this concentration, hyphal elongation is considerably inhibited. 6) The concentration of 10 ppm inhibits hyphal elongation of rice sheath blight fungus.

The above compounds have the effect of inducing the formation of phytoalexins in rice as the active ingredients of Samples 1 to 7 shown in Table 1, and were found to be effective as the active ingredients of rice disease control agents. Was.

Example 3 Production of PO8 and PO9 (1) Culture of rice blast fungus 200 g of peeled potato was cut into small pieces, and 1 L of distilled water was put in the autoclave at 121 ° C. for 60 minutes. After the heat treatment, the potato was filtered off with gauze to prepare a 1 L filtrate. 2% saccharose and 0.5% in the filtrate
Was added to prepare a PSY medium. 200 ml of this medium was dispensed into a 500 ml Erlenmeyer flask and
After sterilization at 21 ° C. for 40 minutes and cooling, rice blast fungus (Race 031 strain) was inoculated. Culture is performed at 26 ° C and 150 rpm.
m was performed for 7 days.

(2) Extraction and Purification of PO8 and PO9 After completion of the culture, the culture solution was filtered with gauze to collect the cells of rice blast fungus. After adjusting the pH to 10.5 by adding distilled water of about 5 times the weight of the cells, ethyl acetate having the same volume as that of water was added, followed by stirring and extraction. The ethyl acetate layer was separated, and the ethyl acetate was distilled off under reduced pressure to obtain an oily residue. 85% residue
The sample solution dissolved in ethanol was used for TSKgel ODS
It was injected into a 120 A column (21.5 mm × 375 mm, manufactured by Tosoh Corporation) and eluted with 91% ethanol. PO8
Was eluted at a retention time of about 25 minutes, and PO9 was eluted at a retention time of about 30 minutes. Each fraction was collected and rechromatographed on the same column. PO8 is a retention time of about 60 minutes in 81% ethanol, and PO9 is a retention time of 40% in 86% ethanol.
Eluted around the minute. Furthermore, each fraction is collected and T
SKgel ODS120T column (21.5 mm ×
375 mm, manufactured by Tosoh Corporation) and fractionated with 95% acetonitrile.
Was eluted at a retention time of about 60 minutes. When the respective fractions were collected and the solvent was distilled off, pure PO8 and PO9 were obtained.

Example 4 Induction of Phytoalexin by PO8 and PO9 (1) Production of Phytoalexin Phosphate buffer containing 0.1% of Tween 20 (manufactured by Wako Pure Chemical Industries) containing PO8 and PO9 obtained in Example 1. Equilibrium solution (20 mM, pH
6.5) to prepare a 50 ppm sample solution.
The sample solution of each concentration and the solvent solution containing no sample are applied to rice (variety: Akitakomachi) grown in a pot,
The activity of inducing phytoalexin production was measured. In this case, the application site was the tip portion of the sixth leaf that was completely developed, and 20 μl (per leaf) of each sample solution was applied drop-wise to the 10 points at appropriate intervals by a capillary pipette.

(2) Extraction of Phytoalexin After the rice treated with the sample solution was cultured in an artificial weather chamber for 7 days, eight treated leaves were cut, cut into pieces, and 5 ml of ethyl acetate was added to 0.1 ml of 0.1 ml.
5 ml (pH 10) of a normal sodium carbonate solution was added, and the mixture was shaken overnight. The ethyl acetate phase was separated and concentrated to dryness, and the residue was dissolved in 0.4 ml of ethanol.

(3) Analysis by HPLC 0.6 ml of 0.02 N hydrochloric acid 1 was added to this solution, mixed, and centrifuged.
1 was subjected to analysis by high performance liquid chromatography (HPLC). HPLC conditions are as follows. Column: TSK-gel ODS 120T (4.6 mm × 300 mm, manufactured by Tosoh Corporation) Solvent: acetonitrile (45): water (55) (volume ratio) Flow rate: 1.2 ml / min Temperature: 50 ° C. Detector: UV 280 nm ( (Phytokasan) / 215 nm (momilactone)

The following Table 2 shows the amount of phytoalexin induced. As is clear from the results in Table 2, the PO8 and PO9 of the present invention have an activity to induce phytoalexins (phytocasans A, B, C, D, momilactone A, B) in rice at a high level of production. It has been found.

[0043]

[Table 2]

Example 5 Rice Blast Infection Protection Test (1) Method Rice (variety: Akitakomachi) seeds were placed in a pot filled with cultivated soil.
Sow 8 seeds per pot, grow until the 6th leaf development stage, 1 ward is 1
Two pots were prepared and two sections were subjected to the test. PO9 was dissolved at a concentration of 50 μg / ml in 20 mM phosphate buffer (pH 7.0) containing 0.1% Tween 20, and 30 ml of the solution was sprayed on 12 pots of rice. As a control, 0.1% Tween 2 was used.
Twelve pots of rice were sprayed with 30 ml of 20 mM phosphate buffer at pH 7.0 containing 0. After leaving at room temperature for 4 hours, the leaves were dried and cultivated in an artificial weather chamber. Seven days after cultivation, 30 ml of a spore suspension of the rice blast fungus race 007 strain (affinity strain) was sprayed on the rice leaf surface in each section, and then humidified.
The inoculation treatment was carried out by allowing to stand in the dark for 24 hours. afterwards,
After being transferred to an artificial weather room and cultivation, 5 days later, the number of infected leaves and the number of uninfected leaves in each section were counted, and the incidence of blast was compared.

(2) Results As a result, in the control plot sprayed with the phosphate buffer solution containing no sample, the number of uninfected leaves was 119, the number of infected leaves was 119, and the disease incidence was 78%. As a result, the number of uninfected leaves was 89 and the number of infected leaves was 65, and the disease incidence was 42%. Approximately the same results were obtained for PO8 that was performed separately.

Example 6 A solution was prepared by mixing a rice blast control agent PO8 (or PO9) and other components in the following mixing ratio by a conventional method. PO8 (or PO9) ───────────────50 μg / ml Tween 20 (manufactured by Wako Pure Chemical Industries) ────────────1000 ppm potassium phosphate buffer Liquid (20 mM, pH 7.0) ────100 ml

Example 7 A liquid formulation was prepared by blending PO8 (or PO9) with other components in the following proportions to control rice wilt disease. PO8 (or PO9) ───────────────50 μg / ml Tween 20 (manufactured by Wako Pure Chemical Industries) ────────────1000 ppm potassium phosphate buffer Liquid (20 mM, pH 7.0) ────100 ml

[0048]

Industrial Applicability The present invention relates to a method for screening a substance that induces the production of phytoalexin in rice, and a rice disease control agent comprising a specific substance having an action of inducing the production of phytoalexin in rice as an active ingredient. According to the present invention, the following effects can be obtained. 1) An elicitor that induces the production of phytoalexin in rice can be screened simply and with high precision. 2) A method using rice phytoalexin phytocasan and momilactone A as an indicator substance for screening an elicitor that induces the production of phytoalexin in rice, and a method for analyzing the same are provided. 3) A rice disease controlling agent comprising a specific substance having an action of inducing the production of phytoalexin in rice as an active ingredient is provided. 4) Phytoalexin has antibacterial properties against rice blast fungus and rice sheath blight fungus. Therefore, the substances selected by the screening method of the present invention are rice disease such as rice blast disease controlling agent and rice sheath blight controlling agent. It is useful as an active ingredient of a controlling agent. 5) A method for producing cerebroside compounds PO8 and PO9 is provided. 6) It is possible to provide a low-toxic, non-polluting, non-polluting rice disease controlling agent which is not only effective in suppressing the occurrence of rice blast and rice sheath blight but also has no residual toxicity.

[Brief description of the drawings]

FIG. 1 shows an infrared absorption spectrum of a cerebroside compound PO8 according to the present invention.

FIG. 2 shows an infrared absorption spectrum of the cerebroside compound PO9 according to the present invention.

FIG. 3 shows ^one of cerebroside compounds PO8 according to the present invention.
1 shows an H-NMR spectrum.

FIG. 4 shows ^one of cerebroside compounds PO9 according to the present invention.
1 shows an H-NMR spectrum.

FIG. 5: ¹³ of cerebroside compound PO8 according to the present invention
¹ shows C-NMR spectrum ¹ .

FIG. 6 shows cerebroside compound PO9 ^{13 according to the} present invention.
¹ shows C-NMR spectrum ¹ .

FIG. 7 shows an infrared absorption spectrum of phytocasan EL according to the present invention.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI A01N 43/60 A01N 43/60 47/18 101 47/18 101B // A01G 7/00 603 A01G 7/00 603 (72) Invention Person Nagahiro Ogasawara 1962 Sone, Nishikawa-machi, Nishikanbara-gun, Niigata Prefecture Inside the Plant Defense System Research Institute Co., Ltd. (56) References JP-A-8-259407 (JP, A) JP-A-7-274752 (JP, A) Hei 5-331016 (JP, A) JP-A-60-190800 (JP, A) Patent 2668200 (JP, B2) Patent 2780737 (JP, B2) (58) Fields investigated (Int. Cl. ⁶ , DB name) A01N 65/00 A01N 35/06 A01N 43/16 A01N 43/36-43/60 A01N 47/18 A01G 7/00 603 CA (STN)

Claims (4)

(57) [Claims] 1. A method for screening an elicitor that induces the production of phytoalexin in rice, comprising using a rice seedling as a test plant, applying a test sample to an appropriate site of the rice seedling, An elicitor screening method, comprising screening an elicitor using a specific phytoalexin produced in a plant as an indicator substance. 2. A test sample is applied drop-wise to the tip of the rice leaf of a young rice plant, and phytoalexin produced in the plant is extracted with a solvent, and phytocasan and momilactone A of rice phytoalexin are used as indicator substances. 2. The method according to claim 1, wherein the analysis is performed by high performance liquid chromatography (HPLC).
The method for screening for the elicitor according to the above. 3. A 2-pyrazinecarboxylic acid, picolinic acid, 2,6-pyridinedicarboxylic acid, 7,3-pyridine having an action of inducing the production of phytoalexin in rice screened by the screening method according to claim 1. A rice disease controlling agent comprising, as an active ingredient, one or more substances selected from pyridinedicarboxylic acid, pyrrole-2-carboxylic acid, oxonic acid, cerebroside compound P08 or P09. 4. A method for producing a cerebroside compound PO8 or PO9 having an action of inducing the production of phytoalexin in rice, wherein rice blast fungus is cultured and extracted from the cells with an organic solvent.