JPH0912504A - New phytocassane el and rice disease controlling agent - Google Patents

Abstract

PURPOSE: To obtain the subject new compound having activity derivatizing production of phytoalexin into rice and having antimicrobe against rice blast disease and sheath blight by adding fungal cell extract of rice blast disease to a liquid culture solution of rice callus. CONSTITUTION: This phytocassane EL is obtained by adding a fungal cell extract of pathogenic fungus of rice blast disease or potato late blight to liquid culture solution of rice callus to produce phytocassane EL of the formula and separating the resultant substance. The substance has the following properties. Color and appearance, colorless gum-like substance; solubility, soluble in acetone, chloroform, methanol and ethanol and dissolvable in water at a concentration of about 100ppm; molecular weight, 316.2062.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel diterpene compound phytocasan EL having a phytoalexin-inducing activity extracted from a rice body. More specifically, the present invention relates to a phytoalexin compound for rice. Phytocasan EL which has an activity of inducing the production of rice and also has an antibacterial activity against rice blast and rice sheath blight, a method for producing the same, and a low-toxicity and pollution-free rice disease control method without worrying about so-called residual toxicity It relates to the agent. Since phytoalexins have strong antibacterial activity against these disease-causing bacteria, application of phytocasan EL of the present invention to rice can prevent infection with rice blast and rice sheath blight.

[0002]

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, which the present inventors have found,
There are C and D (Japanese Patent Application No. 7-43520).

[0003] Substances that induce the production of phytoalexins in plants are called elicitors (Keen, NT; Science).
187: 74-75 (1975)), many substances have been isolated from plant pathogens. As a representative elicitor,
Phytophthoramegasperma f. Sp.glycin as a polysaccharide substance
hepta-β-D-glucopyranoside (Shar
p, JK, B. Valentand, P. Albershim; J. Biol. Che
m. 259: 11321-11336 (1984)), Monilini as a protein substance
a Nicolas A isolated from a fructicola (Cruickshan
k, IAM and DR Perrin; Life Sci. 7: 449-458
(1968)), eicosapentaenoic acid isolated from Phytophthora infestans as a lipid (Bostock, RM, J. Kuca
nd RA Laine; Science 212: 67-69 (1981)).

[0004] In addition to elicitors derived from pathogenic bacteria, certain pesticides, antibiotics, heavy metals and the like are known to have elicitor activity. Components in the rice body such as phytocasan EL have elicitor activity. Things have never been known. As described above, the elicitor induces the production of phytoalexin, which exhibits antibacterial properties against diseased bacteria in plants, and thus can be a highly safe plant disease controlling agent by a different action from conventional synthetic pesticides. However, there are few examples of practical application.Therefore, in the art, a highly safe plant disease controlling agent utilizing such a phytoalexin production inducer has been developed. There was a strong demand for development.

[0005]

Under these circumstances, the present inventors have conducted various searches for substances capable of producing and inducing phytoalexin in the rice body in view of the above-mentioned prior art. It has been newly found that one of the components has such activity. They also found that this substance has antibacterial properties against pathogenic bacteria and is efficiently produced by callus culture of rice. Furthermore, this substance was isolated by means such as solvent extraction and column chromatography, and its structural analysis was carried out. As a result, it was revealed that this substance was a novel substance phytocasan EL having the following structure. The present invention has been completed by clarifying that the substance is effective as an agent for controlling rice blast and rice sheath blight.

[0006]

Embedded image

An object of the present invention is to provide a novel phytocasan EL which is an elicitor which induces the production of phytoalexin in rice and which has antibacterial properties against rice blast fungus and rice sheath blight fungus. is there.

Another object of the present invention is to provide a method for producing phytocasan EL by collecting phytocasan EL from a callus culture of rice.

Another object of the present invention is to provide an agent for controlling rice blast and rice wilt which can control rice blast and rice wilt by applying phytocasan EL to rice. It is assumed that.

[0010]

Means for Solving the Problems The present invention for solving the above-mentioned problems has the following structural formula which has an activity of inducing the production of phytoalexin in rice and has an antibacterial activity against blast fungus and sheath blight fungus. Fight Casan EL, represented by
It is about.

[0011]

Embedded image

[0012] The present invention for solving the above-mentioned problems also comprises adding a cell extract of a plant pathogen such as rice blast or potato blight to a liquid culture of rice callus to produce phytocasan EL. And a method for producing the phytocasan EL described above.

Further, the present invention for solving the above-mentioned problems provides a rice blast disease comprising the above-mentioned phytocasan EL as an active ingredient,
The present invention relates to a rice crop wilt disease control agent.

The present inventors aimed to search for a substance that produces and induces phytoalexin, and applied a sample to the leaf surface of rice, cultivated it in a timely manner, and then produced phytoalexin in the rice body. In the process of conducting various tests to measure the amount of phytoalexin, a solvent extract from a liquid culture of a rice callus to which a cell extract of a plant pathogen such as rice blast or potato blight has been added. High activity was observed. Therefore, the active ingredient (phytocasan EL) can be produced, for example, by adding a cell extract of a plant pathogen such as a rice blast fungus or a potato late blight fungus to a liquid culture of a rice callus and culturing it in a timely manner. Yes, and then Fight Kasan EL
Was produced, and the culture was extracted with a water-insoluble organic solvent such as ethyl acetate, chloroform, ethyl ether, etc., and purified by high-performance liquid chromatography, thin-layer chromatography, or other means. It can be isolated as a single component by treatment.

As a liquid medium of rice callus used in the present invention, any medium conventionally used for tissue culture of rice, which comprises a carbon source, a nitrogen source, inorganic salts, vitamins, plant hormones and the like can be used. However, preferably, for example, DK medium and the like are exemplified. In these media, carbohydrates such as sucrose are used as a carbon source, potassium nitrate, ammonium sulfate, calcium chloride and the like are used as inorganic salts, and aspartic acid, glutamine and the like are used as a nitrogen source. Further, as the vitamins, nicotinic acid, thiamine hydrochloride, pyridoxine hydrochloride and the like are used. As the plant hormone, 2,4-D, kinetin, abscisic acid, indoleacetic acid and the like are appropriately used.

The variety and tissue site of the rice used are not particularly limited, but preferably, for example, callus prepared from Koshihikari seeds is preferably used. In order to form a rice callus, tissue culture of rice may be performed according to a conventional method, and it is not particularly limited. For example, preferably, for example, endosperm and embryo of rice seed excluding rice husks are preferably used. Using a site, this was added to the liquid medium twice as much as 2,4-D,
It may be aseptically cultured in a medium from which aspartic acid and glutamine have been removed.
Rice calli are formed by culturing the cells in the dark at 30 ° C. for 30 days.

In the present invention, phytocasan EL is induced by adding a cell extract of rice blast fungus or potato late blight fungus to a liquid culture of rice callus. Preferable methods for preparing a cell extract component of blast fungus and potato late blight fungus which are inducers of phytocasan EL are, for example, as follows. That is, each strain is inoculated into a rye liquid medium (containing a water extract of 60 g of rye seeds in 1 L, 20 g of saccharose, and 2 g of yeast extract), and shaken at 28 ° C. for 5 to 7 days for rice blast. Culture, and in the case of a potato late blight, cultivate at 18 ° C. for one month. Each culture is filtered to separate the cells. 180 ml of water is added to about 50 g of the cells, ground with a homogenizer, crushed by ultrasonic waves, and thermally decomposed in an autoclave (121 ° C., 60 minutes). The supernatant obtained by centrifuging the digest is used as a phytocasan EL inducer sample. The method for preparing the cell extract component is as follows:
It is needless to say that the present invention is not limited to the above method, but may be used as long as it has the same effect.

The above-mentioned Phytocasan E was added to the callus culture medium.
An L inducer sample is added and cultured for several days, and the supernatant is separated. As a specific process for purifying Phytocasan EL, for example, after extracting the supernatant with a solvent such as ethyl acetate, the extracted component is subjected to TSKgel ODS1.
20A (Tosoh Corporation), ODS120T (Tosoh Corporation)
Examples of suitable methods include purification and isolation by a purification process such as fractionation by high-performance liquid chromatography (HPLC), concentration and drying to dryness, etc., but not limited thereto, and other similar purification means. Can be appropriately combined, and the purification process is not particularly limited.

The phytocasan EL of the present invention is a novel substance and has the following properties. 1) Phytokasan EL is a colorless gum-like substance, soluble in acetone, chloroform, methanol and ethanol, and dissolved in water at a concentration of about 100 ppm. 2) The precise molecular weight of phytocasan EL measured by high-resolution mass spectrometry was 316.2620 (calculated as C ₂₀ H ₂₈ O ₃ , 316.265). 3) Phytocasan EL shows the infrared absorption spectrum shown in FIG. 4) Fight Ca San EL is shown in FIG. 2 ¹ H-NMR
The spectrum is shown. 5) Phytokasan EL is ¹³ C-NMR shown in FIG.
The spectrum is shown. 6) Phytocasan EL shows the CD spectrum (Circular dichroism, circular dichroism) shown in FIG. 7) Phytocasan EL has the property of inducing phytoalexin production in rice. The phytoalexins to be derived include phytocasans A, B, C, D, momilactone A, B and the like. Because these substances have strong antibacterial activity against rice blast fungus and rice sheath blight fungus,
Rice that has been induced with these substances is considered to be resistant to diseased bacteria. 8) Phytocasan EL has an effect of inhibiting germination of spores and elongation of hyphae of rice blast fungus. Phytokasan EL, which inhibits spore germination by 50%, has a concentration of 7 ppm.
And at this concentration the elongation of the hypha is considerably inhibited. 9) Phytocasan EL inhibits hyphal elongation of rice sheath blight at a concentration of 10 ppm.

The phytocasan EL of the present invention has the property of inducing the production of phytoalexin, an antibacterial substance in rice, the germination of spores and the elongation of hyphae of rice blast fungi, as shown in the examples described later. Phytokasan EL is useful as an active ingredient of a rice blast disease controlling agent and a rice wilt disease controlling agent because it has a property of inhibiting the germination of hyphae of the rice wilt fungus. Fight Casan E
Since L has the above-mentioned various properties, it is possible to suppress the occurrence of rice blast and rice sheath blight by applying the compound to rice as a drug in an appropriate form. 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 Examples described later, and the form, formulation means and the like are not particularly limited. Not something.

As a method for applying the compound to rice, as described in Example 5 described later, for example, the compound is dissolved in a 20 mM phosphate buffer solution containing 0.1% Tween 20 at pH 5.5. Then, a method of spraying and spraying the solution on rice is exemplified as a suitable one, but is not limited thereto.
Other methods may be used, and the form of the drug for applying the compound to rice, its use form, the method of application, and the like are not particularly limited.

The phytocasan EL of the present invention is a natural substance derived from rice, is easily decomposed by healthy plant tissues, and has almost no residual properties. Its usefulness is extremely remarkable as a so-called low toxicity, non-polluting plant disease controlling agent which is expected to be developed from the viewpoint of conservation.

[0023]

EXAMPLES The present invention will be described in more detail with reference to the following examples.
The present invention is not limited to the following examples. Example 1 Production of Phytokasan EL 1) Propagation of rice callus Rice calli (cultivar: Koshihikari) was transferred to a DK medium (s
ucrose 30g, KNO ₃ 0.809g, (N
_{H 4) SO 4 0.066g, NaH} 2 PO 4 · 2H 2
O 0.312 g, CaCl ₂ .2H ₂ O 0.148
_{g, MgSO 4 · 7H 2 O} 0.246g, Fe-ED
TA 0.02 g, vitamins 0.101 g, glycine 2 mg, aspartic acid 0.7 g, glutamine
0.7g, 2,4-D 1mg, and as other _{salts, MnSO 4 · 4~6H 2 O,} ZnSO 4 · 7H
_{2 O, CuSO 4 · 5H 2} O, Na 2 MoSO 4 · 2H
Liquid medium containing ₂ O and the required amount of H ₃ BO ₄ )
Swirl culture under conditions of 5.8 and 25 ° C (90 rpm, 25
The cells were grown at 3,000 lux). An appropriate amount of this callus was taken, finely crushed with a spatula, and only the callus passing through a 20-mesh wire net was sieved.

2) Production of Phytokasan EL The sieved callus was placed in a 500 ml Erlenmeyer flask, 90 ml of DK medium was added thereto, and cultivation was performed at 25 ° C. for 2 days at 90 ° C. (90 rpm, 25 ° C., 3000 lux). After that, 1 ml of a solution of a bacterial extract of potato late blight, which had been aseptically filtered, was added to the culture solution, and swirling culture was further continued for 4 days.
After culturing, centrifuge the culture solution (11,500 r
pm, 2 hr) The supernatant was separated.

3) Extraction and Purification of Phytokasan EL The supernatant was adjusted to pH 10.7 by adding sodium carbonate, and then the same amount of ethyl acetate was added thereto to add Phytokasan EL.
Was extracted, and the extract was concentrated to dryness. The residue was dissolved in ethanol, and this was used as a sample solution, followed by separation and purification by two-stage high performance liquid chromatography (HPLC). That is, as the first step, the sample solution was TSKge
l ODS-120A column (21.5 mm x 375
mm, manufactured by Tosoh Corporation) and eluted with 55% acetonitrile (10 ml / min). The phytocasan EL fraction having a retention time of about 41 minutes was collected, concentrated and dried, and the residue was dissolved in ethanol. Next, as a second step, this sample solution was injected into a TSKgel ODS-120T column (21.5 mm × 375 mm, manufactured by Tosoh Corporation) and eluted with 50% acetonitrile (10 ml / m 2).
in). Fight Kasan EL with a retention time of around 46 minutes
The fractions were collected and concentrated to dryness to obtain a single phytocasan EL. About 3 from 1 L of culture supernatant
mg of Phytokasan EL was obtained.

Example 2 Induction of phytoalexin by phytocasan EL 1) Production of phytoalexin Phosphate buffer containing 0.1% of Tween 20 (Wako Pure Chemical Industries) containing phytoalexin EL obtained in Example 1 Solution (20 mM,
pH 5.5) to prepare sample solutions of 10, 20 ppm. The sample solution of each concentration and the solvent solution containing no sample were applied to rice (cultivar: Koshihikari) cultivated in a pot, and 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 of each sample solution (per leaf) was applied dropwise 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 for 7 days in an artificial weather room,
One leaf was taken, shredded, and 5 ml of ethyl acetate and 5 ml of 0.1 N sodium carbonate solution (pH 10) were added thereto, followed by shaking overnight. The ethyl acetate layer was separated and concentrated to dryness, and the residue was dissolved by adding 0.4 ml of ethanol.

3) Analysis by HPLC 0.6 ml of 0.02 N hydrochloric acid was added to this solution, mixed, centrifuged, and 100 μl of the obtained supernatant was
Was subjected to analysis by high performance liquid chromatography (HPLC). The HPLC conditions are as follows. Column: TSK-gel ODS 120T (4.6m
mx 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 (phytocasan), 215
nm (momilactone)

The following Table 1 shows the amount of the induced phytoalexin. As is clear from the results in Table 1, the phytocasan EL of the present invention was obtained by adding phytoalexins (phytocasans A, B, C, D, phytocasan EL, momilactones A and B) to rice at a high production amount. It was found to have an inducing activity.

[0030]

[Table 1]

Example 3 Inhibition test of mycelial growth of blast fungus 1) Method A spore of blast fungus (Pyricularia oryzae race 007) was suspended in water containing 100 ppm of Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a suspension. , 25 μl thereof was placed on a whole object glass plate. The plate was cultured at 28 ° C. for 6 hours to germinate spores. Separately, a sample of Fightkasan EL was added to Tween 20 at 500 ppm.
It was dissolved in water containing it at a predetermined concentration. An aqueous solution 2 of phytocasan EL of a predetermined concentration is added to the spore suspension that has germinated on the plate.
500ppm of sample group and Tween 20 to which 5 μl was added
A control group to which 25 μl of the water was added was prepared, and each group was cultured overnight at 28 ° C., and the elongation state of hypha in each group was observed with a microscope.

2) Results As a result, it was found that in the sample group containing phytocasan EL, the elongation of the mycelium of the blast fungus was inhibited, and that the phytocasan EL had an effect of inhibiting the elongation of the mycelium of the blast fungus. In addition, the concentration of phytocasan EL whose hypha length was about 50% of that in the control group was 7 ppm.

Example 4 Inhibition Test of Hyphal Elongation of Sheath Blight Fungus 1) Method 3.9 g of a commercially available potato dextrose agar medium (manufactured by Eiken Chemical Co., Ltd.) was dissolved in 100 ml of water, and this agar medium solution was tested in 2 ml portions. Dispense into tubes and at 121 ° C,
After sterilizing in an autoclave for 15 minutes, it was cooled to 60 ° C.
Separately, a sample of Fightkasan EL is 500 for Tween 20.
After dissolving to a predetermined concentration in water containing ppm, sterile filtration was performed.
The solution of Phytokasan EL was added to the solution of the agar medium, mixed uniformly, and poured into a petri dish.
An agar plate containing 0 ppm was prepared. Also, as a control, a considerable amount of 500 pp containing no Phytokasan EL.
A blank plate was prepared by adding a Tween 20 aqueous solution to an agar medium. A small piece of an agar medium on which Rhizoctonia solani was cultured was placed at the center of the plate of these petri dishes, cultured at 28 ° C., and the elongation of mycelia was observed 40 hours later. If there is no inhibition, the hypha elongates around the central inoculum and forms a circular flora.

2) Results As a result, it was found that in the sample containing Phytocasan EL, the growth of hypha of Sheath blight fungus was inhibited, and that Phytocasan EL had the effect of inhibiting the growth of hypha of Sheath blight fungus. In addition, no elongation of mycelia was observed in the phytocasan EL 10 ppm section, while the diameter of the microflora of the control plate was 18 mm.

Example 5 Rice Blast Infection Protection Test 1) Method 8 seeds of rice (cultivar: Koshihikari) seeds were sown in a pot filled with cultivation soil, and each seed was grown until the 6-leaf development stage. 2 wards were used for the test. Phytocasan EL was dissolved in phosphate buffer (20 mM, pH 5.5) containing 0.1% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) to a concentration of 35 ppm to prepare a sample solution. 50 ml each of a phosphate buffer solution containing 0.1% Tween 20 and a sample solution was sprayed on each of the rice plants and allowed to stand at about 23 ° C. for 24 hours to dry the leaves.
On this leaf surface, the blast fungus Pyricularia oryzae race 0
The spore suspension of the 07 strain (affinity strain) was sprayed on each section in an amount of 50 ml, and then left under a condition of humidification and darkness for 24 hours to inoculate. After that, move to artificial climate room and cultivate
Days later, the number of gray spots observed on the inaba leaves was counted (18
The average number of lesions per pot in 144 pots of rice) and the degree of blast disease were compared.

2) Results As a result, the average number of lesions per rice plant was 2.17 in the phosphate buffer-treated group containing 0.1% Tween 20, whereas the phytocasan EL sample was In the solution-treated group, the number was 0.91, and the effect of phytocasan EL on suppressing the blast onset was clearly recognized.

Example 6 Rice Blast Disease Control Agent Phytokasan EL and other components were mixed in the following mixing ratio to prepare a liquid preparation by a conventional method. Phytokasan EL {35 μg / ml Tween 20 (manufactured by Wako Pure Chemical Industries)} 500 ppm potassium phosphate buffer (20 mM , PH 5.5) --- 100 ml

Example 7 Rice sheath blight disease control agent Phytokasan EL and other components were mixed in the following mixing ratio to prepare a liquid preparation by a conventional method. Phytokasan EL {35 μg / ml Tween 20 (manufactured by Wako Pure Chemical Industries)} 500 ppm potassium phosphate buffer (20 mM , PH 5.5) --- 100 ml

[0039]

Industrial Applicability The present invention relates to a phytocasan EL having an elicitor activity for inducing the production of phytoalexin in rice, and having an antibacterial activity against rice blast fungus and rice sheath blight fungus. According to the present invention, the following effects can be obtained. (1) A novel diterpene compound that induces phytoalexin production in rice and has antibacterial activity against rice blast fungus and rice sheath blight fungus. (2) An efficient manufacturing method of phytocasan EL is provided. (3) Phytokasan EL induces the production of phytoalexin in rice and has antibacterial properties against rice blast fungus and rice wilt disease. Therefore, the compound is used as a rice blast disease controlling agent and rice wilt disease. It is useful as an active ingredient of disease control agents. (4) It is possible to provide a low-toxic, non-polluting rice disease control agent which is not only effective in suppressing the occurrence of rice blast and rice sheath blight but also has no problem of so-called residual toxicity.

[Brief description of the drawings]

FIG. 1 shows an infrared absorption spectrum of phytocasan EL of the present invention.

FIG. 2 shows a ¹ H-NMR spectrum of phytocasan EL of the present invention.

FIG. 3 shows a ¹³ C-NMR spectrum of phytocasan EL of the present invention.

FIG. 4 shows a CD spectrum (Circular dichroism, circular dichroism) of phytocasan EL of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:91) (72) Inventor Nagahiro Ogasawara 1962, Sone, Nishikawa-cho, Nishikanbara-gun, Niigata Prefecture Plant Defense System Laboratory

Claims (4)

[Claims] 1. The following structural formula having an activity of inducing the production of phytoalexin in rice and having an antibacterial activity against blast fungus and sheath blight fungus. Fightkasan EL represented by. 2. A method for producing a phytocasan EL by adding a cell extract of a plant pathogen such as a rice blast fungus or a potato late blight fungus to a liquid culture of rice callus, and then separating the phytocasan EL. The method for producing the Fightkasan EL according to claim 1. 3. A rice blast disease control agent comprising the phytocasan EL according to claim 1 as an active ingredient. 4. An agent for controlling rice wilt disease comprising the phytocasan EL according to claim 1 as an active ingredient.