KR20220101420A - Composition comprising moriniafungin for controlling plant disease - Google Patents

Abstract

The present invention relates to a composition for controlling plant storage diseases comprising moriniafungin as an effective component. Specifically, the present invention relates to: a composition for controlling plant diseases caused by fungi of the genus Rhizopus (Rhizopus Spp.), comprising moriniafungin as an effective component; and a composition for controlling plant diseases caused by fungi of the genus Rhizopus (Rhizopus Spp.), comprising an ethyl acetate extract of a methanol extract of a Setosphaeria rostrata strain culture or a solvent fraction for the ethyl acetate extract as an effective component.

Description

Composition comprising moriniafungin for controlling plant disease

The present invention relates to a novel use of moriniafungin having antibacterial activity against plant pathogens, and more particularly, the present invention relates to a composition for controlling plant diseases comprising moriniafungin as an active ingredient.

Plant storage disease is a disease that occurs in agricultural products that are being stored after harvest. If the temperature and humidity are not controlled during storage, it promotes the reproduction of pathogens and causes great damage to the crops after harvest. In general, diseases that occur during transportation after harvest are called transportation diseases, diseases that occur during the distribution process of agricultural products such as markets and retail stores are market diseases, and among market diseases, diseases that occur on vegetables and fruits are called fruit and vegetable diseases.

Currently, domestic horticultural crop production reaches 11 trillion won, but the damage from storage bottles is so severe that about 3 trillion won is lost in the process of distribution and storage after harvest.

In particular, bacterial soft rot is a plant disease that begins to form water-acid spots on plants and fruits and rapidly expands to rot plants and fruits.

The control of soft blight is carried out in various ways, such as chemical control using fungicides, seed control, and the use of resistant varieties. By reducing wounds during harvest, losses due to disease can be reduced, and it is managed by using synthetic disinfectants in the packaging line. A broad-spectrum fungicide such as dichloran has been registered as a storage disease control agent and is also used in various crops and flowers. However, the direct use of these synthetic fungicides in fruits and vegetables is regulated worldwide with respect to residual toxicity, and causes negative perceptions among consumers.

Therefore, in the control of plant diseases, it is necessary to develop a new control agent that is harmless to the human body, environmentally friendly and can effectively prevent the spoilage of stored fruits.

Republic of Korea Patent 10-1816952

Accordingly, an object of the present invention is to provide a composition for controlling plant diseases caused by fungi, which includes moriniafungin as an active ingredient.

Another object of the present invention is an ethyl acetate extract for the methanol extract of the Setosperia lostrata strain culture; Or to provide a composition for controlling a plant disease caused by a fungus of the genus Razopus comprising a fraction for the ethyl acetate extract as an active ingredient.

Another object of the present invention is moriniafungin; Ethyl acetate extract for methanol extract of Setosphaeria rostrata strain culture; Or a methanol fraction for the ethyl acetate extract; it relates to a plant disease control method caused by the Rhizopus spp. fungus, comprising the step of treating the plant.

Therefore, the present invention provides a composition for controlling plant diseases caused by the fungus of Rhizopus spp., comprising moriniafungin as an active ingredient.

In one embodiment of the present invention, the morinia fungin may be isolated from Setosphaeria rostrata.

In one embodiment of the present invention, the Rhizopus spp. fungus is R. stolonifer var. stolonifer or R. oryzae.

In one embodiment of the present invention, the plant disease may be soft blight.

In one embodiment of the present invention, the plant may be an apple, peach, pear, red pepper, Chinese cabbage, radish, carrot, garlic, onion, cucumber, melon, tomato or tangerine.

In addition, the present invention is an ethyl acetate extract for the methanol extract of the Setosphaeria rostrata strain culture; Or comprising a solvent fraction for the ethyl acetate extract as an active ingredient, Rhizopus spp. It provides a composition for controlling plant diseases caused by fungi.

In one embodiment of the present invention, the extract or the fraction may contain moriniafungin.

In one embodiment of the present invention, the fraction is 80-100% (v/v) methanol elution in column chromatography of the ethyl acetate extract for the methanol extract of the Setosphaeria rostrata strain culture It may be a methanol fraction of the ethyl acetate extract fractionated with a solvent.

In one embodiment of the present invention, the Rhizopus spp. fungus is R. stolonifer var. stolonifer or R. oryzae.

In one embodiment of the present invention, the plant disease may be soft blight.

In one embodiment of the present invention, the plant may be an apple, peach, pear, red pepper, Chinese cabbage, radish, carrot, garlic, onion, cucumber, melon, tomato or tangerine.

Further the present invention Moriniafungin (moriniafungin); Ethyl acetate extract for methanol extract of Setosphaeria rostrata strain culture; Or a methanol fraction for the ethyl acetate extract; comprising the step of treating the plant, Rhizopus spp. It provides a method for controlling plant diseases caused by fungi.

The extract of the Morinia fungin compound of the present invention and the Setosphaeria rostrata culture containing the compound has a specific and excellent antifungal activity against a plant pathogen, Rhizopus spp. Rhizopus spp. There is an effect that can be usefully used as a control agent for plant diseases caused by fungi.

1 shows a schematic diagram of a process for isolating a morinia fungin compound according to the present invention.
Figure 2 shows the chromatography of the moriniafungin compound is separated using HPLC.
3 shows the chemical structural formula of the moriniafungin compound according to the present invention.
4 shows the mass spectrum analysis result of the moriniafungin compound according to the present invention.
Figure 5 shows the results of analyzing the control effect of soft blight according to the treatment of the moriniafungin compound of the present invention using apples and pears, where A shows the results for apples and B shows the results for peaches.

The present invention is characterized by providing a novel use of a moriniafungin compound having excellent antifungal activity against plant pathogens derived from natural materials.

Specifically, the present invention is characterized in that it provides a composition for controlling plant diseases caused by Rhizopus spp. fungus, comprising moriniafungin as an active ingredient.

The moriniafungin compound according to the present invention may preferably be used isolated from the Setosphaeria rostrata fungus.

The morinia fungin compound of the present invention was obtained from an extract obtained by using a solvent in a culture of Setosperia lostrata, and was obtained by a method comprising the step of separating and purifying the extract by column chromatography. The compound was obtained. At this time, the solvent that can be used to obtain the extract is not limited thereto, but is selected from the group consisting of water, C1 to C4 lower alcohol, hexane, chloroform, methylene chloride, ethyl acetate, acetone and acetonitrile. Any one or a mixture thereof may be used as a solvent.

In an embodiment of the present invention, a crude methanol extract was obtained from a culture containing a culture medium and a culture medium of Setosperia lostrata using methanol, and then the crude methanol extract was again used as an ethyl acetate extract using ethyl acetate. After obtaining , the ethyl acetate extract was loaded on column chromatography to obtain a methanol fraction with a concentration gradient of the methanol elution solvent.

Then, as a result of analyzing the antifungal activity against plant pathogens for each methanol fraction, it was found that the fractions of 80% and 100% methanol elution solvent had excellent antifungal activity. HPLC separation was performed to isolate and identify the moriniafungin compound of the present invention.

Accordingly, the present inventors confirmed the antifungal activity against plant pathogens with respect to the moriniafungin compound isolated and identified in the present invention through MIC analysis. As a result, the moriniafungin compound was It was specifically shown to have excellent antifungal activity.

Specifically, various plant pathogens A. brassicicola, A. oryzae, B. cinerea, C. gloeosporioides, C. orbiculare, F. oxysporum f. sp. lycopersici, R. oryzae, or R. stolenifer var. After the conidia suspension of stolonifer was treated with the moriniafungin compound, their growth inhibitory activity was confirmed as the minimum inhibitory concentration (MIC). R. oryzae, or R. stolenifer var belonging to the genus Rhizopus spp. . It was found to have excellent antifungal activity specifically against stolonifer.

Therefore, through these results, the present inventors found that the morinia fungin compound can be used as an agent for controlling plant diseases caused by Rhizopus spp. fungus.

On the other hand, the plant pathogen, Rhizopus fungus, mostly exists as a saprophytic, and exists as a parasite in a storage organ of a plant. Pathogen spores present in the air germinate after invasion through wounds, and use pectin-degrading enzymes to decompose the pectin material in the middle wall of plant cells, which weakens the binding force between plant cells and causes plant storage diseases such as rot. . In fruits, the initial symptom is that the pericarp around the infected area becomes soft, and as the disease progresses, white mycelium grows on the lesion area, fluid leaks from the infected tissue, and the disease spreads to surrounding healthy fruits. In particular, it occurs when the humidity in the storage is high, and when the disease conditions are good, the disease spreads rapidly and causes great damage within a short time.

In one embodiment of the present invention, in order to confirm that the morinia fungin compound of the present invention can effectively inhibit plant storage disease on fruits, after making a wound on peaches and apples, morinia funzin is dispensed on the wounded area Then, the plant pathogen was inoculated, and after a certain time elapsed, the diameter of the lesion and the condition of the fruit were visually observed.

As a result, the lesion diameter was significantly reduced in the fruit treated with morinia fungin, compared to the untreated fruit, and the occurrence of lesion sites such as softness was suppressed. was found to be superior to the group treated with tebuconazole, which is used as a pesticide.

Therefore, the present inventors found that the morinia fungin of the present invention can be used as a better plant disease control agent that can replace conventional pesticides.

Therefore, the present invention can provide a composition for controlling plant diseases caused by the fungus of Rhizopus spp., which includes moriniafungin as an active ingredient.

In addition, the plant for the purpose of controlling plant diseases in the present invention is not limited thereto, but may be apple, peach, pear, red pepper, Chinese cabbage, radish, carrot, garlic, onion, cucumber, melon, tomato or tangerine.

Furthermore, the present invention provides an ethyl acetate extract for the methanol extract of the Setosphaeria rostrata strain culture; Or, it may provide a composition for controlling plant diseases caused by fungi, comprising a solvent fraction for the ethyl acetate extract as an active ingredient.

As described above, the present inventors have studied to discover a new formulation for controlling plant diseases that is safe and excellent in antifungal activity from nature. Moriniafungin was isolated and identified, and prior to that, in order to isolate the compound, antifungal activity was analyzed for extracts and solvent fractions of the Setosperia lostrata fungus.

As a result, it was found that 80% and 100% methanol fractions had high antifungal activity among the fractions obtained by loading the ethyl acetate extract for the methanol crude extract of the mold Setosperia lostrata on column chromatography.

Moriniafungin is contained in the or the fraction according to the present invention, and the fraction is 80-100 in column chromatography using an ethyl acetate extract for a methanol extract of a Setosphaeria rostrata strain culture. It may be a methanol fraction of the ethyl acetate extract fractionated with % (v/v) methanol elution solvent.

The active ingredient included in the composition for controlling plant diseases according to the present invention may be included in the composition for controlling plant diseases at a concentration of 0.01 to 100 μg/ml. The concentration represents the concentration contained in the composition when treating a plant disease-inducing plant, its seeds or its habitat, and is not limited to the concentration range. It can be appropriately controlled in consideration of the type of plant pathogen, the degree of occurrence, the environment, and the like.

The composition of the present invention may further include substances contained in commonly used pesticides or fungicides, and in addition to the active ingredient, a pharmaceutically acceptable solid carrier, liquid carrier, liquid diluent, liquefied gas diluent, solid as an excipient. It may further comprise a surfactant, such as a diluent, or other suitable adjuvant, for example an emulsifying, dispersing or foaming agent. The control composition in which the active ingredient and the excipient are mixed can be formulated into various formulations known in the agrochemical field, and any formulation method commonly used in the agrochemical field can be used for formulation.

The composition of the present invention may be preferably formulated in wettable powders, granules, powders, emulsions, sprays, emollients, capsules, and gels.

Further the present invention Moriniafungin (moriniafungin); Ethyl acetate extract for methanol extract of Setosphaeria rostrata strain culture; Or a methanol fraction for the ethyl acetate extract; comprising the step of treating plants, the genus Rhizopus (Rhizopus spp.) may provide a method for controlling plant diseases caused by fungi.

The plant disease control method of the present invention is to control by applying the ethyl acetate extract or the methanol fraction for the ethyl acetate extract to the methanol extract of Morinia fungin, Setosperia lostrata strain culture so as to be in direct contact with plant pathogens. , a method of treating the target plant directly is preferred.

Hereinafter, the present invention will be described in detail by way of Examples. However, these Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

<Example 1>

Isolation and identification of moriniafungin compounds having antifungal activity against plant pathogens from Setosphaeria rostrata

Setosphaeria rostrata F3736 fungus was isolated from wood fragments collected in Gaeunsan, Seoul in April 2016. Isolation of the fungus was performed by a general plant-derived fungal isolation method known in the art, and the rDNA sequence of the internally transcribed spacer region was identified using KUC (Korea University Culture Collection), and the fungus corresponds to Setosphaeria rostrata. It was confirmed that

S. rostrata F3736 mold was inoculated into PDA (BD Difco, Franklin Lakes, NJ, USA) and cultured at 25°C for 7 days, and the cultured cells and medium were extracted using methanol. The methanol extract was filtered, concentrated, dried, resuspended in distilled water, and extracted again by adding the same volume of ethyl acetate.

Then, the ethyl acetate extract was separated by Diaion HP-20 gel column chromatography (Mitsubishi Chemical, Tokyo, Japan), and a methanol solvent of a step-wise concentration gradient (0%, 20%, 40%, 60% 80%, 100%) was eluted using , and washed with 500 ml of acetone.

The antifungal activity of each fraction obtained was evaluated by the paper disk diffusion method (8 mm in diameter; Advantec, Toyo, Japan) to evaluate the antibacterial activity against R. oryzae, and 1 mL of R in 100 ml of molten PDA (0.7% agar) oryzae (10 ⁷ conidia/ml) suspension was added to prepare an agar medium in which spores were dispensed. After loading each of the fractions on a paper disk, the spores of the pathogen (R. oryzae) were placed on the PDA medium, and the active ring was observed at 28°C for 2 days.

Thereafter, fractions (80% methanol and 100% methanol fractions) showing inhibitory activity against pathogens (R. oryzae) were collected on a J′sphere ODS-H80 column (250 × 10 mm, 4 μm; YMC Co., Ltd., Kyoto, Japan) equipped with a Varian HPLC system (Agilent Technologies, Santa Clara, CA, USA) was used for further purification. Chromatography was performed for 20 minutes using a 20-95% concentration gradient methanol solvent, followed by isocratic elution with a 95% methanol solvent at an elution rate of 1 ml/min for 20 minutes. The eluate was monitored by absorbance measurement at 230 nm, and fractions showing a peak were collected, and antifungal activity against R. oryzae was analyzed, and the fraction having antifungal activity was designated as Compound A.

Thereafter, the molecular weight of Compound A having antifungal activity was analyzed by electrospray ionization mass spectrometry using ultra-high performance liquid chromatography-tetrapole time-of-flight mass spectrometry. Nuclear magnetic resonance spectra for Compound A were analyzed using a Varian 500-MHz scholarly resonance spectrometer (Varian, Palo Alto, CA, USA) equipped with a 5-mm AutoX DB probe. In addition, ¹ H- and ¹³ C-NMR analysis of compound A was measured in chloroform-d (Cambridge Isotope Laboratory, Andover, MA, USA). Chemical shifts were referenced to the solvent peaks (δH 7.26 ppm and δC 77.0 ppm), and the structure of compound A was determined from spectroscopy-based data. The mass spectrum results of the compound A are shown in FIG. 4, and the ¹ H- and ¹³ C-NMR analysis results are shown in Table 1 below.

As a result of the analysis through the above experiment, the fraction having antifungal activity against R. oryzae was dried, and the active peak (retention time; 14.5 min) was separated and purified through reverse-phase HPLC analysis using a C18 column, NMR and mass spectrometry As a result of analyzing the structure of the identified compound, it was confirmed that it was a moriniafungin compound having the structural formula of FIG. 3 . In addition, a schematic diagram of the process of identifying the moriniafungin compound of the present invention is shown in FIG. 1 , and the result of confirming the active peak through the HPLC analysis is shown in FIG. 2 .

<Example 2>

Analysis of antifungal activity of moriniafungin compounds against various plant pathogens

The antifungal activity against various plant pathogens of the moriniafungin compound isolated and purified from S. rostrata F3736 was performed by analysis of the minimum inhibitory concentration (MIC) value. For this purpose, with various plant pathogens, A. brassicicola, A. oryzae, B. cinerea, C. gloeosporioides, C. orbiculare, F. oxysporum f. sp. lycopersici, R. oryzae, or R. stolenifer var. A conidia suspension of stolonifer (4 × 10 ⁵ conidia/ml) was added to 96-wells along with 25 μl of quadruple-strength potato dextrose broth (PDB; BD Difco). Then, the moriniafungin compound was added to each well to have a final concentration of 0.01, 0.02, 0.03, 0.06, 0.13, 0.25, 0.50, 1, 2, 4, 8, 16, 32, and 64 μg/ml, After incubation at 28ºC for 2 days, the MIC (Minimum inhibitory concentrations) of the moriniafungin compound against plant pathogens was measured.

As a result, as shown in Table 2 above, Colletotrichum spp. The minimum inhibitory concentrations against bacteria were 1 and 8 μg mL ^-1 , respectively, and the minimum inhibitory concentrations against Rhizopus spp. were 0.125 and 0.03125 μg mL ^-1 . On the other hand, antibacterial activity was not shown even at the highest concentration against other phytopathogenic strains.

Therefore, through these results, the present inventors found that, among various plant pathogenic fungi, the morinia fungin compound of the present invention has an excellent antifungal activity specifically against fungi of the genus Rhizopus spp. This means that the moriniafungin compound can be used to control plant diseases caused by fungi of the genus Rhizopus spp.

<Example 2>

Analysis of the soft disease control effect of moriniafungin compound

Peach and pear were used to confirm the effect of the morinia fungin compound in preventing rot by Rhizopus. A solution containing moriniafungin and tebuconazole, known as an antifungal agent, was prepared, respectively, and the solution was 0.05% Tween-20 (v/v) and a compound by concentration (0.04, 0.20, 1, 5, 20ug/ mL) was dissolved in water. Thereafter, the surfaces of apple and peach fruits were washed with 1% NaOCl, and three wounds were made per fruit using a sterilized tip. 5 μL of the solution containing moriniafungin and tebuconazole (positive control material) was dispensed to the wounded area and dried for 1 hour. After 1 hour, the pathogen R. stolonifer var. Spores of stolonifer and R. oryzae were harvested with PDB containing 0.05% Tween 20 and then inoculated at a concentration of 1 × 10 ⁶ spores per 1 ml, 5 μL each. R. stolonifer var. After inoculating stolonifer on peaches and R. oryzae on apples, they were placed in a plastic container filled with water to maintain humidity. After storage at 28°C for 4 days, the lesion diameter was measured.

As a result, as shown in FIG. 5 , the group treated with the moriniafungin compound of the present invention showed a decrease in the lesion site of softness in both apples and pears compared to the group not treated. Specifically, in the case of apples, at a concentration of 25 μg mL ^-1 of moriniafungin, no development of soft disease was observed around the R. Symptoms of softness were observed. R. stolonifer var. In peach fruits inoculated with stolonifer , the progression of soft disease was reduced by 56.5-80% when moriniafungin was treated at a concentration of 0.04-5 μg mL ^-1 , and decreased by 90% at a concentration of 25 μg mL ^-1 . Furthermore, the effect of moriniafungin was found to be superior to that of tebuconazole.

Through the above results, the present inventors showed that the moriniafungin compound isolated and purified from S. rostrata F3736 had excellent antifungal activity against phytopathogenic fungi, and in particular, as it showed excellent antifungal activity specifically for rhizopus genus fungi, , it was found that the moriniafungin compound can be usefully used as a control agent for plant storage diseases caused by fungi of the genus Rhizopus, including soft blight.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

A composition for controlling plant diseases caused by fungi, comprising moriniafungin as an active ingredient. According to claim 1,
The morinia funzin is a composition for controlling plant diseases caused by fungi, characterized in that it is isolated from Setosphaeria rostrata. According to claim 1,
The Rhizopus spp. fungus is R. stolonifer var. A composition for controlling plant diseases caused by fungi, characterized in that stolonifer or R. oryzae. According to claim 1,
The plant disease is characterized in that the soft blight, Rhizopus spp. A composition for controlling plant diseases caused by fungi. According to claim 1,
The plant is an apple, peach, pear, pepper, Chinese cabbage, radish, carrot, garlic, onion, cucumber, melon, tomato or tangerine, characterized in that, Rhizopus spp. Plant disease control caused by fungi for composition. Ethyl acetate extract for methanol extract of Setosphaeria rostrata strain culture; Or comprising a fraction for the ethyl acetate extract as an active ingredient,
Rhizopus spp. A composition for controlling plant diseases caused by fungi. 7. The method of claim 6,
The extract or the fraction is Moriniafungin (moriniafungin), characterized in that it contains, Rhizopus spp. A composition for controlling plant diseases caused by fungi. 7. The method of claim 6,
The fraction is
It is a methanol fraction of the ethyl acetate extract obtained by fractionating the ethyl acetate extract for the methanol extract of the Setosphaeria rostrata strain culture with 80-100% (v/v) methanol elution solvent in column chromatography. Characterized in, Rhizopus spp. A composition for controlling plant diseases caused by fungi. 7. The method of claim 6,
The Rhizopus spp. fungus is R. stolonifer var. A composition for controlling plant diseases caused by fungi, characterized in that stolonifer or R. oryzae. 7. The method of claim 6,
The plant disease is characterized in that the soft blight, Rhizopus spp. A composition for controlling plant diseases caused by fungi. 7. The method of claim 6,
The plant is an apple, peach, pear, pepper, Chinese cabbage, radish, carrot, garlic, onion, cucumber, melon, tomato or tangerine, characterized in that, Rhizopus spp. Plant disease control caused by fungi for composition. Moriniafungin; Ethyl acetate extract for methanol extract of Setosphaeria rostrata strain culture; or a methanol fraction for the ethyl acetate extract;
A method for controlling plant diseases caused by Rhizopus spp. fungus.

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