KR101780096B1 - Selective medium for Fusarium solani comprising radicicol - Google Patents

Abstract

The present invention relates to a selective medium of Fusarium solani containing radicicol exhibiting antifungal activity and its use. The medium containing radicicol of the present invention effectively inhibits the growth of pathogenic fungi other than pseudomonas solani, but does not affect the growth of fusarium solani, and thus can be used as a selective medium for fusarium solani. In addition, the selective medium can easily detect fungal solani within a short time, and can be useful for diagnosing a plant disease caused by fungal solani.

Description

Selective medium for Fusarium solani < RTI ID = 0.0 > radicicol < / RTI >

The present invention relates to a selective medium of Fusarium solani containing radicicol exhibiting antifungal activity and its use. More particularly, the present invention relates to a selective culture method, a detection method, and a diagnostic method of ginseng root rot disease using a selective medium containing radicicol.

Fusarium solani is a filamentous fungus that is distributed worldwide and is often separated from soil or plant debris. Fusarium solani is known as a pathogen of various plant diseases and is known to cause root rot of ginseng which is mainly grown in East Asia including Korea. However, since many plant pathogenic fungi are found together with other pathogenic fungi other than pseudomonas solani, it is necessary to use natural environments and diseased plants for accurate diagnosis and pathological analysis of plant diseases as well as studies on fusarium solani It is necessary to selectively isolate and cultivate the fungal solani.

On the other hand, radicicol is a secondary metabolite found from Cylindrocarpon radicicola (Evans, G. 1965. The antibiotic activity of Cylindrocarpon radici-cola . University of Sydney. Ph.D. (Schulte, TW, et al., 1998). Antibiotic radicicol binds to the N-terminal domain of Hsp90 and Cell Stress Chaperon 3: 100-108). Radicicol is known to have anticancer, antimicrobial activity and so on and is being used in various pharmaceutical compositions. For example, Korean Patent Laid-Open Publication No. 2014-0021520 discloses a pharmaceutical composition comprising radicicol and a derivative thereof, which is useful for treating diseases mediated by kinase and HSP90, such as autoimmune diseases, inflammatory diseases, Neurodegenerative or neurodegenerative diseases, cancer, cardiovascular diseases, allergies, asthma, or hormone-related diseases. Brefeldin A, another secondary metabolite secreted by fungal strains, is a lactone antibiotic and is known to inhibit protein transport from the endoplasmic reticulum to the Golgi apparatus (Klausner, RD, et al. 1992. Brefeldin A: insights into the control of membrane traf-fic and organelle structure. J. Cell Biol. 116: 1071-1080). However, the use of these compounds as selective media for pseudomonas solani has not yet been known.

It is an object of the present invention to provide a selective medium capable of selectively separating and cultivating fungal solani using radishcorn.

It is another object of the present invention to provide a method for culturing or detecting fuararium solani using a selective medium containing radicicol.

It is still another object of the present invention to provide a method for diagnosing a plant disease mediated by Fusarium solani using a selective medium containing radicicol.

In order to achieve the above object, the present invention provides a selective medium of Fusarium solani containing radicicol.

The selection medium may be prepared by adding an effective amount of radishcor to the base medium that can be used for culturing the fungal solani. There is no restriction on the type of the "basic medium" as long as it can be used for culturing the fungal solani. In one embodiment of the present invention, the base medium may be a liquid or a solid medium. In one embodiment of the present invention, the basal medium may be a medium such as MM (minimal medium), PDA (potato dextrose agar), CM (complete medium), CDA (czapek dox agar) broth, or CMC (carboxylmethyl cellulose) medium. The "effective amount" refers to an amount of radishcor contained in the selective medium which indicates antibacterial and / or antifungal activity against a microorganism other than fucaria solani contained in the sample. Depending on the culture condition, the medium and the kind of pathogen An amount that is most suitable for selectively culturing the fusarium solani can be selected. In one embodiment of the present invention, the concentration of radishcor added to the selective medium may be 50 mg / L or higher, but is not limited thereto.

In one embodiment of the present invention, the selective medium may further include a substance exhibiting antibacterial or antifungal activity against a microorganism other than fungal solani. In one embodiment of the present invention, the material may be Brefeldin A.

In one embodiment of the invention, the selective medium exhibits antimicrobial or antifungal activity against fungi, including fungi, preferably fungal, more preferably acaricides, incomplete fungi and egg fungi, other than puarium solani . In one embodiment of the present invention, examples of the pathogenic fungus in which the selective medium exhibits antifungal activity include Rhizoctonia solani, Botrytis cinerea, Alternaria panax, Sclerotinia sclerotiorum, and Pythium ultimum. However, the present invention is not limited thereto.

In addition, the present invention provides a method for selectively culturing a fungus solani using the selective medium. The culturing method includes a step of inoculating and culturing the sample in the selective medium.

In one embodiment of the present invention, the sample may be one separated from ginseng, preferably from ginseng caught in root rot disease of ginseng or its surrounding soil. In one embodiment of the present invention, the sample is selected from the group consisting of Rhizoctonia solani, Botrytis cinerea, Alternaria panax, Sclerotinia sclerotiorum, and Pythium ultimum. However, the present invention is not limited thereto.

In one embodiment of the present invention, the culturing is preferably performed at 25-30 DEG C, more preferably at 25 DEG C, but is not limited thereto.

The present invention also provides a method for detecting pseudomonas solani using radishcorn.

In one embodiment of the present invention, the detection method includes a step of inoculating and culturing a sample in a medium containing radicicol. The sample is a sample for confirming whether or not the presence of fusarium solani is present. In one embodiment of the present invention, the sample may be separated from ginseng, and the detection method may be a method for identifying or isolating the fusarium solania present in ginseng. In one embodiment of the present invention, the sample is selected from the group consisting of Rhizoctonia solani, Botrytis cinerea, Alternaria panax, Sclerotinia sclerotiorum, and Pythium ultimum. However, the present invention is not limited thereto.

In one embodiment of the present invention, the culturing is preferably performed at 25-30 DEG C, more preferably at 25 DEG C, but is not limited thereto.

In addition, the present invention provides a method for diagnosing a plant disease mediated by Fusarium solani using the above detection method.

In one embodiment of the present invention, the diagnostic method comprises the steps of inoculating and culturing a sample in a medium containing radicicol; Isolating the fungi growing in the medium; And confirming whether the separated fungus is fusarium solanii.

In one embodiment of the present invention, the plant disease may be ginseng root rot disease, and the sample may be one isolated from ginseng or surrounding soil that is caught in ginseng root rot disease. In one embodiment of the present invention, the step of determining whether the isolated fungus is Fusarium solanii may be performed by a genetic method including morphological analysis and / or nucleotide sequence analysis.

The medium containing radicicol of the present invention effectively inhibits the growth of pathogenic fungi other than pseudomonas solani, but does not affect the growth of fusarium solani, and thus can be used as a selective medium for fusarium solani. In addition, the selective medium can easily detect fungal solani within a short time, and can be useful for diagnosing a plant disease caused by fungal solani.

FIG. 1 shows the results of comparing the mycelial growth of Fusarium solani and other pathogenic fungi to determine the optimal culture temperature of Fusarium solani (cultivation at 10, 15, 20, 25 and 30 ° C, respectively). RS: Rhizoctonia solani , Fusarium solani , BC: Botrytis cinerea , Gray mold fungal pathogen, AP: Alternaria lyophilus, a fungus pathogen, Rhizoctonia solani , Alternaria panax , SS: sclerotinia sclerotiorum , PU: Pythium ultimum , a root- rot fungus .
FIG. 2 is a photograph showing the effect of the medium containing radishcorn on mycelial growth of fungal solani and other pathogenic fungi according to an embodiment of the present invention (cultivation at 15 and 20 ° C, respectively). FS: F. solani , RS: R. Solani , BC: B. Cinerea , AP: A. Panax , SS: S. Sclerotiorum , PU: P. Ultimum .

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Experimental strain

Soil pathogenic fungi were isolated from ginseng roots collected from Korean negative. Fusarium solani strains were purified by single spore isolation, and the Rhizoctonia solani strains were subcultured from growing boundaries. Each strain was identified based on morphological features and phylogenetic analysis including nutritional growth and conidia production.

From the mycelium cultured in CM (complete medium) medium according to the known method (Chi, MH, et al, 2009. A quick and safe method for fungal DNA extraction, Plant Pathol, J. 25: 108-111) and genomic DNA was extracted. The internal transcribed spacer (ITS) of each strain was PCR-amplified according to the known method (White, TJ, et al., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press Inc. New York, USA) The amplified product was purified and sequenced. The analyzed sequences were compared with the sequences registered in the NCBI database, the Agricultural Genetic Resources Information Center (Suwon, Korea) and the Plant Pathogenic Fungi Genetic Resource Bank (Seoul, Korea), and Rhizoctonia solani , Identify strains of Fusarium solani , Alternaria panax , Sclerotinia sclerotiorum , Pythium ultimum and Botrytis cinerea strains. Respectively.

≪ Example 2 >

Measurement of mycelial growth rate of experimental strains

Mycelial agar plugs (5 mm in diameter) were inoculated into PDA (potato dextrose agar), MM (minimal medium) and CM medium (Leslie et al., USA) to compare the mycelial growth rate of the fungus solani and other fungal strains Growth rates were measured every 24 hours while culturing at 10, 15, 20, 25, and 30 ° C for 4 days, respectively, at the culture temperatures of 10, 15, 20, . Experiments were performed in triplicate and Tukey tests were performed using SPSS 12.0 software (SPSS Inc., Chicago, USA) to evaluate the significance (P <0.05) between the mean values of each sample.

As a result of the experiment, it was confirmed that the pseudomonas solani grown at a culturing temperature of 20 ° C or lower was similar to or slower than other fungi, but grown at a temperature of 25 ° C or higher (Fig. 1) . In addition, it was confirmed that the mycelial growth of the fungi except for the sclerotinia cleperothorum and pisumi ulthhimma was drastically reduced at a culture temperature of 15 ° C or lower. These growth patterns according to the incubation temperature were common regardless of the kind of medium (PDA, MM and CM medium). Taken together, these results indicate that the optimum temperature condition for selective cultivation of fusarium solani is 25 to 30 ° C.

&Lt; Example 3 >

Selective antimicrobial activity of radishcorn medium

To investigate whether the test strains were resistant to radicicol and brefeldin A, the MM medium supplemented with various concentrations of radishcor and / or brepeldin A (Sigma Aldrich) The strains were inoculated. After autoclaved autoclaved, the above compounds dissolved in methanol were added to the chilled medium at 50 占 폚. Plates were cultured at 10, 15, 20, 25, and 30 캜 after inoculating each of the fungi into the medium and mycelial growth was measured every 48 hours for 8 days after inoculation.

As a result, mycelial growth of fungi other than Fusarium solanii was slightly inhibited by 50 mg / L of brespendin A, and addition of 50 mg / L of radishcor completely inhibited all temperature conditions . Similar results were obtained in the medium supplemented with both compounds. FIG. 2 is a photograph taken on day 4 after inoculation of experimental strains into medium supplemented with 50 mg / L of radepsilon and / or 50 mg / L of radishcor and incubation at 15 ° C and 20 ° C, respectively .

These results show that radicicol inhibits the growth of other fungal species except fosarium solani, and is a component that can effectively isolate stomach strains.

In addition, in order to investigate the effect of radicicol on the germination of conidia, we used PDB (potato dextrose broth) and CMC (carboxylmethyl cellulose) medium (Capellini, et al., 1965. Macroconidium formation in submerged cultures by Conidium spores (10 ⁵ conidia / ml) of each of the pseudomonas sp. produced in the nonsporulating strain Gib-berella zeae. Mycologia 57: 962-966 were added to 50 g / L radishcor with or without 50 mg / ml &lt; / RTI &gt; MM medium. The germination rate of conidia was measured every 2 hours after inoculation while culturing at a temperature of 20 ° C for 24 hours. As a result, the effect of radishcorp was not observed as a whole.

Claims (13)

Preparing a ginseng root sample comprising Fusarium solani , Alternaria panax , and Sclerotinia sclerotiorum ;
Isolating a fungal strain from the sample; And
And culturing the isolated fungal strain in a medium containing radicicol. The method according to claim 1,
The culture may be performed by selectively culturing Fusarium solani from the group consisting of Fusarium solani , Alternaria panax and Sclerotinia sclerotiorum . &Lt; / RTI &gt; The method according to claim 1,
Characterized in that the medium further comprises Brefeldin A. The method according to claim 1,
Characterized in that the medium is added to the MM, PDA, CM, CDA, PDB or CMC medium. 5. The method of claim 4,
Wherein the radicicol is contained at a concentration of 50 mg / L. delete delete delete delete delete delete delete delete

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