JPH08259407A - Production of antimicrobial terpene compound - Google Patents

Abstract

PURPOSE: To obtain a partially new antimicrobial terpene compound in high yield by producing an antimicrobial terpene compound by adding the microbial extract derived from plant pathogenic fungi to a rice callus culture fluid followed by isolating the compound. CONSTITUTION: A rice callus culture fluid is spiked with an microbial extract derived from plant pathogenic fungi such as rice neck rot or potato disease fungi as a phytocassane or momilactone-inducing substance to effect producing phytocassane or momilactone as a terpene compound presenting antimicrobial activity on rice neck rot fungi followed by isolating the compound, thus affording the objective new antimicrobial terpene compound, i.e., phytocassane A, B, C or D of formula I (R1 is H or OH; R2 is H, OH or O) or momilactone A of formula II or momilactone B of formula III. The phytocassane A, B, C or D is a naturally occurring substance, being effective on rice neck rot and rice sheath blight and useful as a low-toxic, pollution-free antimicrobial agent. The momilactone A or B, which suppresses rice seed germination, is effective on rice neck rot.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a terpene compound having antibacterial activity against rice blast fungus, etc. by adding a bacterial cell extract of plant pathogenic fungi such as rice blast fungus or potato pathogen to a liquid culture of rice callus. The present invention relates to a method for producing an antibacterial terpene compound, phytocasane or momilactone, at a high production rate by producing phytocasane and momilactone.

[0002]

2. Description of the Related Art Generally, it is known that plants show a resistance reaction (hypersensitive reaction) when contacted with a pathogenic bacterium and produce phytoalexin which exhibits antibacterial activity against the pathogenic bacterium in tissues around the reaction site. There is. Rice phytoalexins include momilactone A, B, oryzalexin A, B,
C, D, E, F, S, sakuranetin, oryzalic acid A, B, oryzalide A, B are known, and other novel compounds, phytocasan A, B, C, found by the present inventors are also known. There is D.

The following are examples of the research results to date concerning rice phytoalexin. 1) Momilactone A, B (formation of phytoalexin momilactone in rice) D. Cartwright, P.M. Langcake,
R. J. Pryce, D.M. P. Leworthy an
d J. P. Ride: Nature, 267, 511
-513 (1977) 2) Oryzalexin A (separation of Oryzalexin A, a novel phytoalexin from rice blast diseased rice leaves). Akatsuka, O .; Kodama, H .; Kat
o, Y. Kono and S.K. Takeuchi: A
gric. Biol. Chem. , 47,445-44
7 (1983) 3) Oryzalexin A, B, C (report of chemical structure, short report) Y. Kono, S. et al. Takeuchi, O .; Kodam
a and T.A. Akatsuka: Agric. Bi
ol. Chem. , 48, 253-255 (1984) 4) Oryzalexin A, B, C (Oryzalexin A,
Separation and properties of B and C, antibacterial activity against rice blast fungus) Akatsuka, O .; Kodama, H .; Sec
ido, Y. Konoand S. Takeuchi:
Agric. Biol. Chem. , 49, 1689-
1694 (1985) 5) Oryzalexin A, B, C (Detailed Report on Structure) Y. Kono, S. et al. Takeuchi, O .; Kodam
a, H.A. SEKIDOAND T. Akatsuka:
Agric. Biol. Chem. , 49,1695-
1701 (1985) 6) Oryzalexin D (separation, structure, activity). Sekido, T .; Endo, R.M. Suga, O.M.
Kodama, T .; Akatsuka, Y. Kono
and S. Takeuchi: J. Pesticid
e Sci. , 11, 369-372 (1986) 7) Oryzalexin E (separation, structure and activity of oryzalexin E, a novel rice phytoalexin from UV-irradiated rice). Kato, O.M. Kodama and T.K. Aka
Tsuka: Phytochemistry 33,7
9-81 (1993) 8) Oryzalexin F (separation, structure, and activity of oryzalexin F, a novel rice phytoalexin from UV-irradiated rice). Kato, O.M. Kodama and T.K. Aka
Tsuka: Phytochemistry, 36, 2
99-301 (1994) 9) Oryzalexin S (separation of Oryzalexin S, a novel rice phytoalexin from UV-irradiated rice, structure). Kodama, W.M. X. Li, S. Tamogam
i and T. Akatsuka: Biosci. B
iotech. Biochem. , 56, 1002-1
003 (1992) 10) Sakuranetin (isolation of flavanone skeleton phytoalexin from UV-irradiated rice and activity against rice blast fungus) O. Kodama, J .; Miyakawa, T .; Aka
tsuka and S. Kiyosawa: Phyto
Chemistry 31, 3807-3809 (19)
92) 11) Oryzalide A (Separation of Oryzalide A, which has antibacterial activity against bacterial wilt of rice leaf blight, from rice cultivar resistant to leaf blight of rice). Watanabe, Y. Sakai, T .; Tera
oka, H .; Abe, Y. Kono, J .; Uzawa,
K. Kobayashi, Y .; Suzukikand
A. Sakurai: Agric. Biol. Che
m. , 54, 1103-1105 (1990) 12) Oryzalide A, B, oryzalic acid A (isolation of oryzalide A, B, oryzalic acid A having antibacterial activity against rice leaf blight fungus from rice leaf disease resistant variety Inaba) ) Y. Kono, J .; Uzawa, K .; Kobayashi
i, Y. Suzuki, M .; Uramoto, A .; Sa
Kurai, M .; Watanabe, T .; Teraok
a, D. Hosokawa, M .; Watanabe a
nd H. Kondo: Agric. Biol. Che
m. , 55, 803-811 (1991) 13) Oryzalic Acid B (Structure of Oryalic Acid B and related A, B, C substances). Watanabe, Y. Kono, M .; Watan
abe, J .; Uzawa, T .; Teraka, D .; H
Osokawa, Y .; Suzuki, A .; Sakura
i and M. Teraguchi: Biosci.
Biotech. Biochem. , 56, 113-1
17 (1992) 14) Osteospermum
m) Seed plant diterpene compound (not phytoalexin, but the same cassan as phytocasan)
e) A compound having a skeleton. However, the structure is different from that of phytokasan, and there is no description about physiological activity.) Ferdinand Bohlmann, Michael
l Wallmyer, Jasmin Jakupov
ic and Jurgen Ziesche: Phy
tochemistry, 22 (7), 1645-16
51 (1983) 15) Phytocasins A, B, C, D (activity against rice blast fungus and rice wilt pathogen, chemical structure) Japanese Patent Application No. 7-43520

Although these phytoalexins have been isolated from rice plants, a method of separating momilactones A and B and oryzalexin A, B, C and D from liquid cultured cells of rice has been studied ( Japanese Patent Application No. 63-4190
3). However, this method directly inoculates the rice blast fungus, does not use the bacterial cell extract of the rice blast fungus or the potato pathogen, and the effectiveness of the bacterial cell extract has not been examined at all. Moreover, the production amount was extremely small, and it was impossible to expect practical use. Phytokasan is a novel compound found by the present inventors, and includes:
C and D can be collected from the resistant reaction site of the rice struck by the rice blast fungus or rice wilt fungus, but there has been no reported case including the production method thereof. Further, according to the research conducted by the present inventors so far, in order to utilize phytocasan as a plant disease control agent, a large amount of rice is cultivated, inoculated with pathogenic bacteria, and resistant reaction sites are collected to collect active ingredients. Had to be separated. However, this method has a problem that a great deal of labor is required for plant cultivation, inoculation work of pathogenic bacteria, etc., and that the separation of the plant component from the effective substance is complicated.

[0005]

In view of the above-mentioned prior art and the research results of the present inventors, the present inventors have focused on a callus liquid culture method for plants, which is often used in the production of plant components, As a result of testing the method of adding a bacterial cell extract of a plant pathogenic fungus such as rice blast fungus or potato pathogen as a phytocasane and fomilactone inducer to the callus culture medium and culturing, a plant such as rice blast fungus or potato pathogen By using the bacterial cell extract of pathogenic bacteria,
The inventors have found that a large amount of phytocasane or momilactone can be easily obtained at a high production rate in a short period of time and completed the present invention.

That is, the present invention is to add phytocasan extract as a phytocasan-inducing substance to a callus culture solution of rice to add phytopathogenic fungal cell extract of phytopathogenic fungi such as rice blast fungus or potato phytophthora bacillus to produce phytocasan. An object of the present invention is to provide a method for producing phytocasan, which is characterized by separation.

In the present invention, a cell extract of plant pathogenic fungi such as rice blast fungus or potato pathogen is added as a momilactone inducer to a callus culture solution of rice to produce momilactone, which is then separated. It is an object of the present invention to provide a method for producing momilactone characterized by the above.

[0008]

The liquid medium for rice used in the present invention is a medium that has been conventionally used for tissue culture of rice and comprises a carbon source, a nitrogen source, inorganic salts, vitamins, plant hormones and the like. Either can be used, but preferably D
K medium. In these media, carbohydrates such as sucrose are used as a carbon source, and inorganic salts and potassium sources such as potassium nitrate, ammonium sulfate, calcium chloride, aspartic acid and glutamine are used. Further, as the vitamins, nicotinic acid, thiamine hydrochloride, pyridoxine hydrochloride and the like are used. As a plant hormone, 2,4-D,
Kinetin, abscisic acid, indoleacetic acid and the like are used as appropriate.

The variety and tissue site of rice to be used are not particularly limited, but callus prepared from Koshihikari seeds is preferable. In order to form rice callus, it may be carried out according to a conventional method and is not particularly limited, but preferably, for example, the endosperm and the embryo part excluding the chaff of rice seeds are treated with the above liquid medium. 2,4-D may be added in a double amount to aseptic acid and glutamine-free medium may be cultivated aseptically, for example, by statically culturing in the dark at pH 5.8 and 25 ° C. for 30 days, rice Callus is formed.

In the present invention, an antibacterial terpene compound is induced by adding a bacterial cell extract of a plant pathogen such as rice blast fungus or potato pathogen to a liquid culture of rice callus. Blast fungus, which is an inducer of phytocasan,
A preferable example of the method for preparing a bacterial cell extract component of a phytopathogenic fungus such as a potato pathogen is as follows, for example. Rye broth (containing 1 g of 60 g of rye seeds in water, 20 g of sucrose, and 2 g of yeast extract in 1 L) was inoculated with each strain, and rice blast fungus was at 28 ° C.
Shake culture for 5 to 7 days, and cultivate potato pathogens at 18 ° C for 1 month. Each culture is filtered to separate the cells. 180 ml of water was added to about 50 g of cells, and the mixture was ground with a homogenizer and sonicated, and then autoclaved (12
Thermal decomposition is performed at 1 ° C for 60 minutes. The supernatant obtained by centrifuging the decomposed product is used as a phytocasan inducer sample. The fomilactone inducer can be prepared in a similar manner. It goes without saying that the method for preparing the bacterial cell extract component is not limited to the above-mentioned method, and can be similarly used if it has the same effect.

After adding the above-mentioned phytocasan and fomilactone inducer to the callus culture medium, the culture is cultured for several days, and the supernatant is centrifuged. The supernatant liquid can be extracted with a solvent such as ethyl acetate, and the solvent can be distilled off to obtain a mixture of crude phytocasane and momilactone. Fight Kasan A, B,
The amounts of C and D components and the amounts of momilactone A and B components are
It can be confirmed by high performance liquid chromatography (HPLC), and each component can be obtained singly by collecting the peaks of the corresponding components. The HPLC conditions for the separation of phytocasan are as follows. Column: TSK-gel ODS 120T (4.6 mm x 300 mm) Solvent: Acetonitrile (45): Water (55) (volume ratio) Flow rate: 1 ml / min Detector: UV 280 nm Further, as for momilactone, it is as follows. . Column: TSK-gel ODS 120T (4.6 mm x 300 mm) Solvent: Acetonitrile (45): Water (55) (volume ratio) Flow rate: 1 ml / min Detector: UV 215 nm

Fightcasins A, B, C and D of the present invention
Is a novel substance, which is a compound having a cassane skeleton represented by the following general formula (I).

[0013]

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Fightcasins A, B, C, D of the present invention
Is a starting material from the supernatant of the above culture, ethyl acetate,
It can be isolated as a single component by extraction treatment with a solvent such as methanol, ethanol or acetone and purification treatment by means of column chromatography, high performance liquid chromatography, thin layer chromatography and the like.

As a concrete process of purification, for example,
After extraction with a solvent, the phytocasane fraction was separated by adsorption and elution treatment by column chromatography on Seppak C-18, and then TSKgel ODS1.
High Performance Liquid Chromatography (H
A method for purification and isolation by a purification process such as fractionation by PLC) and a concentration-drying operation is exemplified as a preferable method, but the method is not limited to the method, and other similar purification means may be appropriately combined and carried out. However, the purification process is not particularly limited.

Phytocasins A, B, C, D of the present invention
Has the following properties. 1) Phytocasins A, B, C, and D are all colorless gum-like substances, such as acetone, chloroform, methanol,
It is soluble in ethanol and 100% in water containing surfactant.
It dissolves at a concentration of around ppm. 2) The precise molecular weight by high resolution mass spectrometry is as follows. Fightcasan A: 316.2052 (calculated value as C ₂₀ H ₂₈ O ₃ 316.2065) Fightcasan B (assuming one hydrogen atom is added):
335.2286 (calculated value as C ₂₀ H ₃₁ O ₄ 335.2223) Phytocasan C: 318.2164 (calculated value as C ₂₀ H ₃₀ O ₃ 318.2133) Phytocasan D: 316.2048 (calculated value as C ₂₀ H ₂₈ O ₃ 316.2065) 3) Phytocasins A, B, C and D show the ¹³ C-NMR spectra shown in FIGS. 4) Phytocasans A, B, C and D have the property of inhibiting spore germination and hyphal elongation of rice blast fungus, and when these are applied to rice, the occurrence of rice blast can be inhibited. . The concentration of phytocasan that inhibits hyphal elongation of rice blast fungus by 50% was 10 pp for phytocasan A.
m, phytocasan B is 3 ppm, phytocasan C is 6 ppm, and phytocasan D is 20 ppm. 5) Phytocasans A, B, C, and D have the property of inhibiting the elongation of hyphae of rice wilt disease fungi, and when these are applied to rice, the occurrence of rice wilt disease can be inhibited. Regarding the concentration of phytocasan that inhibits the growth of mycelia of rice wilt pathogen, phytocasan A was 17 ppm, and the growth of mycelia was significantly inhibited, and phytocasan B was 15 ppm.
When Phytocasan C is 10 ppm, mycelial elongation is completely inhibited. Phytokasan D significantly inhibits hyphal elongation at 50 ppm.

Phytocasins A, B, C, D of the present invention
Has the property of inhibiting the germination of spores of rice blast fungus and the elongation of mycelium, and the property of inhibiting the growth of mycelium of rice wilt disease fungus.These are rice blast disease control agents and rice wilt disease control agents. It is useful as an active ingredient of an agent. Since the phytocasans A, B, C, and D of the present invention have the above-mentioned various properties, by applying the compound to rice as a drug in an appropriate form, the occurrence of rice blast disease and rice blight Can be suppressed. The form of the drug for applying the compound to rice, the form of use, the method of application, etc. are not particularly limited, and for example, based on the concentration that inhibits the elongation of hyphae of rice blast fungus by 50%. An appropriate amount and form of a drug containing the compound as an active ingredient may be prepared and applied by a conventional method.

Phytocasins A, B, C, D of the present invention
Is a natural substance produced in the plant body of rice, and it is easily decomposed by healthy plant tissue and has almost no residue. Therefore, it has high antibacterial activity against rice blast fungus and rice wilt fungus. In addition to having activity, its usefulness is extremely remarkable especially as a so-called low toxicity, non-polluting antibacterial agent which is expected to be developed from the viewpoint of environmental protection.

On the other hand, momilactones A and B are known as substances that suppress germination of rice seeds and show antibacterial activity against rice blast fungus, and the molecular formulas and molecular weights of momilactones A and B are as follows. Shown. Momilactone A C ₂₀ H ₂₆ O ₃ 314 Momilactone B C ₂₀ H ₂₆ O ₄ 330 Momilactones A and B have the following chemical structures.

[0020]

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[0021]

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Momilactones A and B can be purified in the same manner as in the case of phytocasan, using the supernatant of the culture as a starting material. As shown in Examples below, the production amounts of momilactone A and B were 0.245 μg and 0.864 μ / ml of the culture solution according to the conventional method.
However, according to the present invention, 3.3 μg, 10.5
It was μg, and the production amount was remarkably increased, and it was found that the production amount of momilactone, which was conventionally difficult to achieve a high production rate, could be significantly improved.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 (Formation of Callus of Rice) The embryo and endosperm portion of rice (Koshihikari) seeds from which the chaff had been removed were sterilized with a 70% ethanol solution and a 1% sodium hypochlorite solution, and then DK medium was used. 2,4-D is 2 except aspartic acid and glutamine
The cells were planted in a medium containing double volume and 1% of agarose. Callus is induced after 30 days and is transferred to liquid medium.

(Propagation of Callus of Rice) Callus of rice (Koshihikari) was treated with DK medium (1 L, sucrose 30 g,
KNO ₃ 0.809 g, (NH ₄ ) SO ₄ 0.06
_{6g, NaH 2 PO 4 · 2H} 2 O 0.312g, Ca
Cl ₂ · 2H ₂ O 0.148 g, MgSO ₄ · 7H ₂
O 0.246 g, Fe-EDTA 0.02 g, vitamins 0.101 g, glycine 2 mg, aspartic acid 0.7 g, glutamine 0.7 g, 2,4-D 1
mg, other _{salts, MnSO 4 · 4~6H 2 O,} ZnS
_{O 4 · 7H 2 O, CuSO} 4 · 5H 2 O, Na 2 MoO
₄ · 2H ₂ O, liquid medium containing the required amount of H ₃ BO ₃₎
, PH 5.8, 25 ° C, 14 days, swirling culture (90 r
pm, 25 ° C., 3,000 lux) and grown.
Take an appropriate amount of this callus and crush it finely with a spatula.
Only the callus that passed through the wire mesh of the mesh was sieved.

(Production of phytocasan) The screened callus was placed in a 500 ml Erlenmeyer flask, 90 ml of DK medium was added, and vortex culture was carried out for 2 days (90 rpm, 25 ° C., 3, 0).
00 lux), and then 1 ml of a sterile-filtered bacterial cell extract component solution of aseptically filtered potato is added to the culture solution, and further 4
The swirling culture was continued for a day. After completion of the culture, the culture solution was centrifuged (12,000 rpm), and the same amount of ethyl acetate was added to the obtained supernatant solution to extract phytocasan, and the extract solution was concentrated to dryness under reduced pressure. Take an appropriate amount of concentrate and use ethanol 4
As a result of dissolving in 0% liquid and analyzing by high performance liquid chromatography, the amounts of the respective components of phytocasan are as shown in Table 1.

[0026]

[Table 1] Types of phytocasan Production amount of phytocasan μg / ml of culture supernatant fluid phytocasan A 6.3 phytocasan B 0.6 phytocasan C 15.4 phytocasan D 1.2

After fractionating each component of phytosakan by HPLC, the corresponding peak portion was collected and concentrated to dryness. As a result of dissolution in a small amount of acetone and analysis with a gas chromatograph mass spectrometer, mass spectra showing molecular ion peaks corresponding to the respective molecular weights were obtained (FIGS. 5 to 8). The same results were obtained when the same procedure was performed using the extracted components of rice blast fungus.

(Production of Momilactone) The screened callus was placed in a 500 ml Erlenmeyer flask, 90 ml of DK medium was added, and swirling culture was carried out for 2 days (90 rpm, 25 ° C., 3,000).
0 lux), 1 ml of the sterile-filtered bacterial cell extract component of Phytophthora infestans was added to the culture solution, and the swirling culture was continued for another 4 days. After the completion of the culture, the culture solution was centrifuged (12,000 rpm), and the same amount of ethyl acetate was added to the obtained supernatant solution to extract momilactone, and the extract solution was concentrated to dryness under reduced pressure. Take an appropriate amount of the concentrate and add 40 parts of ethanol.
As a result of dissolution in 100% liquid and analysis by high performance liquid chromatography, the amount of each component of momilactone is as shown in Table 2. In addition, the production amount of momilactone A and B produced by the conventional method (method of directly inoculating rice blast fungus) is
Since it is about 0.245 μg / ml and 0.864 μg / ml, it can be seen that the production amount of momilactone according to the present invention has an extremely high production rate as compared with the conventional method.

[0029]

[Table 2] Type of Momilactone Momilactone production amount μg / ml of culture supernatant liquid Momilactone A 3.3 Momilactone B 10.5

After fractionating each component of momilactone by HPLC, the corresponding peak portion was collected and concentrated to dryness. As a result of dissolving in a small amount of acetone and analyzing with a gas chromatograph mass spectrometer, mass spectra showing molecular ion peaks corresponding to respective molecular weights were obtained (FIGS. 9 to 1).
0). The same results were obtained when the same procedure was performed using the extracted components of rice blast fungus.

[0031]

INDUSTRIAL APPLICABILITY Since the method for producing an antibacterial terpene compound according to the present invention uses a liquid medium, tank culture is possible and a large amount of sample can be prepared. In addition, since the supernatant of callus culture solution is used as an extraction raw material, grinding work is unnecessary compared to rice plants, and less pigment is mixed in, so extraction and purification work is facilitated and large-scale sample preparation is possible. It became easy to do. Furthermore, the present invention makes it possible to remarkably improve the production amount of the antibacterial terpene compound by using the cell extract of plant pathogenic fungi such as rice blast fungus or potato pathogen as the inducer.

[Brief description of drawings]

FIG. 1 ^{13 of} Phytocasan A produced according to the present invention
A C-NMR spectrum is shown.

FIG. 2 ^{13 of} Phytocasan B produced according to the present invention
A C-NMR spectrum is shown.

FIG. 3 ^{13 of} Phytocasan C produced according to the present invention
A C-NMR spectrum is shown.

FIG. 4 ^{13 of} Phytocasan D produced according to the present invention
A C-NMR spectrum is shown.

FIG. 5 shows a mass spectrum of Phytocasan A produced according to the present invention.

FIG. 6 shows a mass spectrum of Phytocasan B produced according to the present invention.

FIG. 7 shows a mass spectrum of Phytocasan C produced according to the present invention.

FIG. 8 shows a mass spectrum of Phytocasan D produced according to the present invention.

FIG. 9 shows a mass spectrum of Momilactone A produced according to the present invention.

FIG. 10 shows a mass spectrum of Momilactone B produced according to the present invention.

Front Page Continuation (72) Inventor Nagahiro Ogasawara 1962 Sone, Nishikawa-cho, Nishikanbara-gun, Niigata Prefecture Plant Defense Systems Laboratory, Inc.

Claims (3)

[Claims] 1. A method for producing an antibacterial terpene compound by adding a bacterial cell extract of a plant pathogenic bacterium such as rice blast fungus or potato phytophthora to a liquid culture of rice callus to produce an antibacterial terpene compound, which is then separated. A method for producing an antibacterial terpene compound. 2. The method according to claim 1, wherein the antibacterial terpene compound is phytocasan A, B, C or D. 3. The method according to claim 1, wherein the antibacterial terpene compound is momilactone A or B.