KR101249854B1 - Composition and Method for Controlling Plant Disease Occurred by Magnaporthe grisea Using a Fruit Extract of Pittosporum tobira Ait or Saponin ⅢA3 Isolated from the Fruit Extract - Google Patents

Abstract

The present invention relates to a plant disease control composition by Magnaporte grisea using chaponin ⅢA ₃ which can be isolated from a nectar fruit extract, a fraction of a nectar fruit extract, or a berry tree and a plant disease control method by magnaporte grisea. It starts. Both the extract and the saponin ⅢA ₃ showed antifungal activity against Magnaporte grisea.

Description

Composition and Method for Controlling Plant Disease Occurred by Magnaporthe grisea Using a Fruit Extract of Pittosporum tobira Ait using Donna Fruit Extract or Chaponin IIIA3 Isolated thereon or Saponin ⅢA3 Isolated from the Fruit Extract}

The present invention relates to a composition for controlling plant diseases and a method for controlling plant diseases by Magnaporte grisea using a gingko berry extract or chaponin IIIA3 isolated therefrom.

Magnaporte Griséa (the scientific name of the incomplete generation is Pyricularia grisea ) is a causative agent of schizophrenia and belongs to the fungal scrotum of fungi, and exists mainly as conidia. The colorless conidia are pear-shaped and have two diaphragms. Optimal mycelial growth temperature for pathogens is 27-28 ° C. The time required for intrusion is about 6 hours at 24 ° C and 8 hours at 20 ° C and 28 ° C.

These pathogens overwinter in the form of conidia, scatter, and attach to and infiltrate the host to form lesions. When there is frequent rain, lack of sunshine, cold and humid weather, the soil temperature is low, the soil moisture content is low, and when nitrogen fertilizer is overused. It is a good condition.

The host plants of Magna Forte Grissea are rice plants such as rice, grass, wheat, barley, and blood.

Rice blast is a disease that causes great damage at all stages of growth from the early to late stages of rice production in the world where rice is grown (Leung & Taga, Advances in Pathology Genetic of Plant Pathogenic Fungi , pp. 175-). 188, 1988). The disease occurs under conditions of frequent rain and insolation, and according to the damage area, it is divided into leaf blast, ear blast, ear branch blast, rice blast, node blast, and leaf blast. The cases of young mothers are called fever.

Turf blast occurs mainly in St. Augustinegrass, Kentucky bluegrass, Perennial ryegrass, and tall fescue, and sometimes in Korean grass. Round or oval, pale gray lesions formed on the leaves or stems of grass, and these lesions overlap, causing the entire leaves to brown and die. In 18 samples of 6 golf courses in Jeju Island, 25.87% of the diseases were found. Dallas Spot 20.69%. Turf fever was found in 19.28% (Jeju Environment Daily December 14, 2009 article).

Methods to reduce the blast damage caused by Magna Forte Grisse include the use of resistant varieties, fertilization control, seedling control such as vegetarian density control, chemical control using chemical pesticides such as tricyclazol and Capropamid, and microbial culture such as actinomycetes. extracts such as Patent No. 2006-0019866) or pomegranate peel and leek seeds (the Korean Journal of Mycology , 33 (2): 86-88), turmeric extract (Korean Patent Publication No. 2006-0039954) and the like can be roughly classified into biological control methods.

The present invention has antifungal activity against Magnaporte grisea, the causative agent of febrile disease ( Pittosporum tobira Ait.) Biological control using fruit extract and chaponin ⅢA ₃ isolated from the extract is presented.

An object of the present invention is a Pittosporum tobira Ait.) The present invention provides a composition for controlling plant diseases caused by Magnaporte grisea using fruit extract and chaponin IIIA ₃ isolated from the extract.

Another object of the present invention is to provide a method for controlling a plant disease by Magna Porte Grisse using the composition.

Wherein the present invention is about 80% of the ethanol extract, the extract butanol, etc. fraction and a single substance of Shah ponin Magna Forte so Seah of pathogens such as ⅢA ₃ (saponin ⅢA ₃₎ the rice blast isolated from the fraction of the Pittosporum tobira fruit It is completed by confirming having fungal activity.

Chaponin IIIA ₃ isolated from nectar fruit is a known substance (Joseph D. Connolly et al., Nat . Prod . Rep . , 2005, 22: 230-248; H. D'Acquarica et al., Tetrahedron 2002, 58: 10127-10136), its chemical formula can be found in [Formula 1] below, its IUPAC name is 12-oleanene-3,15,16,28- tetraol-21-angeloyl-22-acetyl-3-O- [β-D-glucopyranosyl- (1 → 2)]-[α-L-arabinopyranosyl- (1 → 3)]-[α-L-arabinofuranosyl- ( 1 → 4)]-β-D-glucuronopyranoside.

The chaponin IIIA ₃ can be separated according to the method disclosed in the above cited document or the method disclosed in the following examples of the present invention. The documents cited above are considered part of this specification.

<Shaponin IIIA ₃ >

In view of the foregoing, the control agent composition of the present invention is characterized in that it comprises a saponin IIIA ₃ which can be separated from the nectar fruit extract, the quince fruit extract, or the nectar fruit extract as an active ingredient.

In addition, in the present specification, "pig tree fruit extract" refers to a pig tree fruit to be extracted, regardless of the extraction method, lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, butanol, acetone, hexane, ethyl acetate, dichloromethane, water or their The extract obtained by extraction with a mixed solvent is meant. Regardless of the extraction method, it is to be understood that the extraction method may be applied to any method such as cooling, refluxing, heating, ultrasonic radiation, and the like, as long as the extraction object is extracted through immersion in the extraction solvent. Nevertheless, the term "donberry fruit extract" means preferably an extract obtained by extracting with water, ethanol or a mixed solvent thereof, more preferably a mixed solvent of water and ethanol, in particular 70-90% ethanol (where% is v / v). The extract may be filtered, concentrated and the phase may be liquid or powder.

In addition, in the present specification, "fractions of bilberry fruit extract" is a bilberry fruit extract, preferably a bilberry fruit extract obtained with a mixed solvent of water and ethanol, in particular 70-90% ethanol, a mixed solvent of methanol and water, particularly 10-40 It means the fraction of any solvent layer obtained by suspending in% methanol, then fractionated with butanol, dichloromethane, ethyl acetate or hexane, preferably the fraction of any solvent layer obtained by fractionation with butanol, furthermore Suspended in a mixed solvent of methanol and water, in particular 10 to 40% methanol, and then sequentially fractionated using hexane, dichloromethane, ethyl acetate and butanol means an extract, preferably a fraction of the butanol layer. By "sequentially fractionating" here is meant that fractions of a mixed solvent of water and methanol are continued to be used after fractionation to fractionate into solvents in the order listed above.

In the present specification, the term "active ingredient" means a component that can exhibit activity alone or in combination with a carrier which is not active in itself.

In addition, in this specification, "control" means the prevention, treatment or alleviation of symptoms of plant diseases caused by Magna Porte Grisse.

In addition, in the present specification, "plant diseases caused by Magna Forte Grisse" means a rice blast and grass blast, and barley, wheat, blood and the like are also host plants of Magna Porte grisea, meaning that they also include blasts of these plants. .

The control agent composition of the present invention may be included in any amount within the range of 1 to 99% by weight of the active ingredients, depending on its formulation, subjects, parts, etc., based on the total weight of the control agent composition of the present invention, It will typically be included in the range of 1 to 30% by weight, usually in the range of 10 to 20% by weight in liquids, and typically in the range of 30 to 40% by weight in powders.

The control composition of the present invention may be prepared as a formulation of the active ingredients, or may be mixed with an agriculturally acceptable carrier to be formulated into an emulsion, solution, suspension, hydrating agent, flow agent, powder, granule, dispersant, and the like.

In the above, the "agrochemically acceptable carrier" is combined with the active ingredient of the control agent composition of the present invention to promote the spreading, storage and / or transport of the active ingredient without inhibiting the activity of the active ingredient and acceptable to plants It refers to any substance that does not exhibit the above toxicity.

Carriers that can be used are soil natural minerals (e.g. kaolin, clay, talc, chalk), soil synthetic minerals (e.g. highly dispersed silica, silicate), carbon based materials (charcoal, bitumen, etc.), sulfur, natural or synthetic Resins, fertilizers, cellulose based materials (sawdust and corncobs), water, aromatic solvents (e.g. Solvesso products, xylene), paraffins (e.g. mineral oil fractions), alcohols (e.g. methanol, butanol, pentanol, Benzyl alcohol), ketones (e.g. cyclohexanone, methyl hydroxybutyl ketone, diacetone alcohol, mesityl oxide, isophorone), lactones (e.g. gamma-butyrolactone), pyrrolidone (pyrrolidone) , N-methylpyrrolidone, N-ethylpyrrolidone, n-octylpyrrolidone), acetates (glycol diacetate), glycols, dimethyl fatty acid amides, fatty acids, fatty acid esters, and mixtures thereof.

The control composition of the present invention may also include one or more adjuvants such as wetting agents, dispersants, antifreezes, thickeners, preservatives, electrodeposition agents.

Such adjuvants may be selected and used appropriately from those known in the art, for example, alkylnaphthalene sulfonate (particularly diisopropylnaphthalenesulfonate or diisobutylnaphthalenesulfonate) may be used as the wetting agent, and nonionic as a dispersant. Dispersants (ethylene oxide / propylene oxide block polymers, alkylphenol polyglycol ethers, tristyrylphenol polyglycols, etc.), anionic dispersants (ligninsulfonic acid, naphthalenesulfonic acid, phenolsulfonic acid, dibutylnaphthalenesulfonic acid, alkylarylsulfonates, alkyl sulfides) Pate, alkylsulfonate, etc.) or mixtures thereof, methanol, ethanol, isopropanol, butanol, glycol, glycerin, diethylene glycol and the like can be used, and as a thickener, cellulose derivatives, polyacrylic acid derivatives, xanthan Modified clay As the preservative, dichlorophene, isothiazolene, benzyl alcohol, etc. can be used, and the electrodeposition agent is polyvinyl alcohol, polyvinylpyrrolidone, polyacrylate, polymethacrylate, polybutene, polyisobutylene , Polystyrene, polyethyleneamine, polyethyleneamide, polyethyleneimine, polyether and the like can be used.

The control composition of the present invention may further contain other known components such as herbicide components, insecticidal components, bactericidal components, etc., depending on the purpose of use, the environment of the arable land, and the like.

In another aspect, the present invention relates to a method for controlling plant diseases by Magna Porte Grisea using the control composition of the present invention as described above.

The method for controlling plant diseases by Magnaporte Grissea of the present invention is characterized by comprising the step of contacting the plant composition of the present invention, such as rice, grass, barley, wheat.

The contacting step will typically be accomplished by spraying on the affected rice, grass, barley, wheat, but by spraying or incorporating into soil, which is the habitat environment of rice, grass, or the like, or by spraying or seeding the seeds or seedlings thereof. Or by spraying or mixing with environmental growth medium or soil.

Alternatively, the step of contacting may be made by mixing with a fungicide, insecticide, herbicide, fertilizer and the like.

The amount of the active ingredient of the present invention's control agent in plants such as rice and grass is comprehensively determined by the extent of growth of rice, grass, etc., degree of disease, soil conditions, rainfall, types of active ingredients to be used, and the like. Will be taken into account. Usually, it is used at 10 to 500 g, preferably 30 to 250 g per hectare, and when spraying and mixing the soil, it is used at 10 to 1000 g, preferably 100 to 500 g per hectare.

As described above, according to the present invention, the composition of the control of plant diseases caused by Magna Porte grisea using the extract of the bilberry fruit and the active substance which can be separated from the extracts, and the plant diseases caused by Magna porte grisea using the composition It can provide a control method of.

Figure 1 is a schematic diagram of the production process of 80% ethanol extract and fractions thereof.
Figure 2 is a result showing the antifungal activity of Magna Forte grisea of 80% ethanol extract and fractions thereof of Prunus.
FIG. 3 shows the results of antifungal activity against Magnaporte griesea of 14 primary purified fractions of the Prunus butanol fraction.
FIG. 4 shows the antifungal activity of Magnaporte Grisea of the 11th highest purified of the 13 secondary tablets obtained from the 12th purified having the highest antifungal activity.
Figure 5 is a schematic diagram of the active material separation process.
FIG. 6 is a 1H NMR spectrum of an active substance, chaponin IIIA ₃ (purification No. 11 in the secondary purification).
Figure 7 is a 13C NMR spectrum of the active substance chaponin IIIA ₃ (purification 11 in the secondary purification).
8 is a DEPT spectrum of an active substance, saponin IIIA ₃ (purification No. 11 in the secondary purification).
9 is an LC-MS / MS spectrum of the active substance chaponin IIIA ₃ (purification 11 in the secondary purification).

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example  1> Wicker  Fruit extract and Fraction  Produce

1 kg of crushed pork tree was put in 80% ethanol, immersed for 48 hours, extracted three times, and the extract obtained by filtration was concentrated under reduced pressure to remove the solvent and lyophilized to obtain 228 g (22.8%) of a powdery extract.

Then, 100 g of the obtained ethanol extract was suspended and dissolved in 100 times of 20% methanol, and then sequentially partitioned into hexane (n-hexane), dichloromethane (CH ₂ Cl ₂ ), ethyl acetate (EtOAc) and butanol (BuOH). 9.46g (9.46%) in hexane layer, 4.42g (4.42%) in dichloromethane layer, 9.71g (9.71%) in ethyl acetate layer, 23.8g (23.8%) in butanol layer and residual water layer (20% methanol solvent) Layer) 52.12 g (52.12%) of a fraction were obtained.

A schematic diagram of the fraction preparation process from the 80% ethanol extract and 80% ethanol extract is shown in FIG.

< Example  2> Wicker  Fruit extract Fraction Magnaporte In greece  Antifungal Activity Assessment for

In order to evaluate the mycelial formation inhibitory ability of anti-fungal mycobacterium extracts and fractions, mycelia of 6 mm diameter at the edges of mycelia were cultured on potato dextrose agar (PDA) for 5 days Sections were collected, respectively, 50 μl / mL of the extract and 40 μl of the fractions were dropped on a 8 mm diameter paper disk, inoculated in potato agar medium, and cultured at 27 ° C. for 7 days to determine the production of the flora.

The results are shown in Figure 2, the most excellent antifungal activity was observed in the butanol fraction of the Pleurotus fraction compared to the 80% ethanol extract (1 and 5, control (80% EtOH); 2, water fraction; 3, 80% ethanol) Extract; 4, butanol fraction; 6, ethyl acetate fraction; 7, dichloromethane fraction; 8, hexane fraction).

< Example  3> Wicker  Fruit Fraction  Active ingredient separation and purification and its Purified Magnaporte In greece  About Antifungal  Activity evaluation

10 g of butanol fractions identified as the most active above were subjected to column chromatography using reverse phase silica gel to obtain a total of 14 primary purified products. The column size was 7 cm × 15 cm and 20, 40, 60, 80, 100% methanol was used as the mobile phase. Antifungal activity of 14 primary tablets was evaluated in the same manner as in <Example 2>. As a result, the antifungal activity of the purified product of No. 12 (PTO-B-12) was Pure silica gel column chromatography was performed on 1.2 g of the purified product No. 12. The column size was 2.5 cm × 50 cm and chloroform: methanol (3: 1, 1: 1, v / v) was used as the mobile phase. The mobile phase was flowed at a rate of 1.5 ml per minute and a total of 13 secondary purifications were obtained.

Similarly, the antifungal activity was evaluated in the same manner as in <Example 2> with respect to the total 13 secondary tablets, and as a result, the activity of the 11th purified product (PTO-B-12-11) in the secondary tablets Was the highest. The antifungal activity of No. 11 secondary tablets is shown in FIG. 4.

The purified product 11 was run on TLC (silica gel 60 F 254, chloroform; methanol = 2: 3, v / v) and Rf was 0.7 and appeared as a single spot.

5 is a schematic diagram showing the separation process of the active material.

< Example  4> Identification of active substances

To analyze the structure of the isolated active material, 20 mg of the purified product was dissolved in methanol-d4 and the NMR spectrum was measured. In the 1 H NMR spectrum (FIG. 6), 10 methyl groups were identified, and one of them was identified as a methyl group (d 2.04 ppm, s) linked to an acetyl group. In addition, it was expected that the glycoside was formed by the signals around 5-3.0ppm, and the presence of the double bond was confirmed by the signal around 5.5-5.8ppm.

13C NMR spectrum (FIG. 7) confirmed the presence of two carbonyl groups (176.3, 169.5 ppm) together with carbonyl carbon (173.0 ppm) of the acetyl group. In addition, two double bonds were identified by signals of 127.0, 129.4, 139.2, and 143.6ppm, and four sugars were expected to be attached as glycosides with four anomer carbons per 108.3, 105.8, 104.2, and 103.0.

In addition, 11 quaternary carbons, 28 CH, 10 CH ₂ , and 10 CH ₃ were identified in the DEPT spectrum (FIG. 8), and the total carbon number of the compound was 59. Thus, H. D'Acquarica et al., Tetrahedron 2002, 58: 10127-10136] identified the structure of the active substance as Saponin IIIA ₃ .

LC-MS / MS analysis was performed to determine the molecular weight of the identified material. Saponin IIIA _{3 had} a molecular weight of 1232.58 and was analyzed to be 1256.12 in sodium-bound form by LC-MS / MS analysis. As a result of performing MS / MS for more accurate structure confirmation, a cleavage pattern unique to Saponin IIIA ₃ was obtained as shown in FIG. 9.

Claims (9)

Plant disease control composition by Magna Porte Gricea comprising chaponin IIIA ₃ as an active ingredient.
delete delete delete delete The method of claim 1,
The composition comprises a pesticide acceptable carrier and at least one auxiliary agent selected from the group consisting of wetting agents, dispersants, antifreeze agents, thickeners, preservatives and electrodeposition agents, plant disease control by Magna Porte Grisce Composition.
The method of claim 1,
The plant disease caused by Magna Porte Grisse is a plant disease control plant composition by Magna Porte Grisse, characterized in that the plant disease affects rice, grass, wheat, barley or blood.
A method for controlling a plant disease by Magnaporte grisea comprising the step of contacting the plant with the plant disease control composition according to any one of claims 1, 6 and 7.
9. The method of claim 8,
The plant is a rice, grass, barley, wheat or blood method of controlling plant diseases by Magna Porte Grisse, characterized in that.

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