KR101873978B1 - COMPOSITION FOR PREVENTING PLANT PATHOGENS COMPRISING EXTRACT FROM SPENT SUBSTRATE OF Lentinula edodes AND METHOD FOR PREPARING THE SAME - Google Patents

Abstract

The present invention relates to a plant disease control agent which contains, as an active ingredient, a concentrate of an extract extracted from a medium after cultivation of Lentinula edodes and which is used for prevention or control of a plant disease including oxalic acid ≪ / RTI > Thus, the present invention can improve the antimicrobial activity by increasing the content of oxalic acid by extracting the composition from the postharvest medium after producing the shiitake, and concentrating the same to improve the antimicrobial activity. In addition, It is possible to manufacture raw materials with little expenditure on raw materials and to easily procure raw materials from farmhouses, thereby reducing production costs and utilizing them as eco-friendly organic farming materials harmless to the human body.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for inhibiting plant diseases, which comprises a medium extract after shiitake fermentation,

The present invention relates to a composition for controlling a plant disease comprising an extract of a medium after shiitake mushroom harvest and more particularly to a concentrate of an extract extracted from a medium after cultivation of shiitake mushroom (Lentinula edodes) And a method for producing the same.

There are fungi, bacteria, and viruses that cause plant disease, among which fungi and bacterial plant diseases account for more than 80%. Most of the control methods depend on chemical methods using pesticides, and there is a need for an effective environment-friendly method due to concerns about drug resistance and environmental pollution. As an environmentally friendly method, a microbial pesticide using an antagonistic microorganism and an antiseptic using a plant-derived natural substance have been developed. A total of 109 species of antimicrobial activity inhibiting the growth of bacteria and fungi have been reported by searching for antibacterial effect against bacteria by using 317 strains of 204 species of Staphylococcus aureus. Antimicrobial substances derived from fruiting body mycelium of fungi have been identified, Most of the related pathogens have been studied. In recent years, Chen et al. Have sought agricultural application of edible mushroom-derived culture filtrate for bacterial plant fungal and plant pathogenic bacteria inhibition. However, in order to apply mycelial culture filtrate to packaging, additional processing costs such as mass culture and extraction system are required, resulting in a problem of lowering economic efficiency.

As an alternative, the use of spent mushroom substrate (SMS) may be considered. It is presumed that the SMS composition contains dense mycelium and a wide variety of physiologically active substances such as various wood degrading enzymes and secondary metabolites. SMS is a solid culture form and it can separate active ingredients only by adding simple extract without culture process and it can be converted into useful resources with low cost and high added value.

Shiitake mushrooms have anticancer, immunological and antioxidant functions, and antimicrobial active substances such as lenthionine have been used from fruiting body and mycelium. The cultivation of highland in Korea is rapidly changing from logging to sawdust cultivation, and about 50% or more of them are now producing 37,000 tons of shiitake mushrooms annually with sawdust cultivation.

Pepper blight caused by pepper is caused by Phythopthora capsici and is the most serious disease which causes the greatest damage to pepper. A similar soil infectious disease is tomato blight caused by the plant pathogenic bacterium Ralstonia solanacearum. These phytopathogenic fungi cause disease in the soil and spray chemical pesticides directly on the soil, which can be a major problem for environmental pollution such as groundwater. It is a plant disease that is difficult to control once it occurs and is environmentally friendly It is necessary to develop a control agent.

Korean Patent Laid-Open Publication No. 10-2013-0134351 Korean Patent Publication No. 10-2015-0055650

DISCLOSURE OF THE INVENTION An object of the present invention is to solve the above problems, and an object of the present invention is to provide a method for producing a shiitake mushroom, The present invention relates to a method for controlling a bacterial plant disease derived from an Asoranaceae room and extracting a composition for promoting plant growth, thereby mass-producing a control composition with a simple process without a separate process facility.

Another object of the present invention is to enhance the antimicrobial activity against plant diseases by increasing the content of oxalic acid by concentrating the extract obtained by extracting the medium remaining after the elevation harvest.

Another object of the present invention is to provide a method for reducing the production cost of a composition for the control of plant diseases by using the remaining medium after the elevation harvest as a raw material, There is.

Another object of the present invention is to utilize a by-product of edible mushroom as a raw material to prepare a composition for controlling plant diseases, and thereby utilize it as an environment friendly organic farm material harmless to human body.

According to an aspect of the present invention,

There is provided a composition for controlling plant diseases, which contains, as an active ingredient, a concentrate of an extract obtained from a medium after cultivation of Lentinula edodes containing oxalic acid, and which is used for preventing or controlling plant diseases do.

The extract may be an extract by hot water extraction.

The concentration of oxalic acid contained in the concentrate of the extract may be 0.6 to 2.0 mg / ml.

The plant bottle may be a plant bottle derived from Phytophthora capsici or Ralstonia solanacearum.

The phytopatholipase-derived plant disease or the plant disease derived from the Ralstonia solanacearum may be any one selected from the group consisting of Solanaceae plants, Cucurbitaceae plants and Fabaceae plants Can be plant bottles for.

The phytophthora capsaicin-derived plant disease may be pepper blight disease, and the plant disease originating from the Ralstonia solanaceae may be a tomato blight disease.

The plant disease controlling composition may further comprise a preservative.

The preservative is selected from the group consisting of Potassium Sorbate, Sorbic Acid, Propyl Gallate, Butylated Hydroxy Toluene and Butylated Hydroxy Anisole It can be either.

According to another aspect of the present invention,

a) preparing the remaining medium after cultivating shiitake mushroom; b) disrupting said medium to produce a disrupted medium; c) hydrothermally extracting the shredded medium to produce a hot-water extract containing oxalic acid; And d) concentrating the hot-water extract to produce a concentrate having an increased content of oxalic acid than the hot-water extract.

The concentrate can be prepared by concentrating the hot-water extract to 15 to 25 times.

After step d), e) adding a preservative to the concentrate.

In step c), the crushed medium is added to the hot water extractor, water is added, and the hot water extract can be prepared by hot water extraction at 70 to 90 ° C for 1 to 3 hours.

The hot-water extract of step c) may have an oxalic acid content of 0.3 to 0.7 mg / ml.

In step c), the weight ratio of the medium and water that has been shredded may be from 1: 5 to 1:10.

The remaining medium after cultivation of the mushroom may be the medium remaining after cultivation of mushroom in solid state fermentation.

The solid medium includes sawdust and a nutrient source, and the nutrient source may include at least one selected from the group consisting of rice bran, wheat bran, beet pulp, corncob, cotton bran, cotton bran, and kraft bran.

The medium remaining after cultivation of the mushroom may be the medium remaining after collecting mushrooms four to eight times in the solid medium.

The present invention relates to a method for producing shiitake mushroom from a post-harvest medium which produces shiitake mushroom, a method for producing shiitake mushroom from a postharvest medium, A composition for controlling bacterial plant diseases derived from stony sauranase room and a composition for promoting plant growth can be extracted and concentrated to produce a composition for control by a simple process without a separate process facility.

In addition, the composition for controlling plant diseases according to the present invention can enhance the antimicrobial activity against plant diseases by increasing the content of oxalic acid by concentrating the extract extracted from the medium after cultivation of Lentinula edodes.

In addition, the present invention can reduce the production cost of the composition for the control of plant diseases due to the fact that the culture medium remaining after the harvesting is used as a raw material, and the raw material can be easily procured from the farm.

In addition, the present invention can be utilized as an eco-friendly organic farm material harmless to the human body by preparing a composition for controlling plant diseases by using a by-product of edible mushroom as a raw material.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flowchart sequentially showing a method for producing a composition for controlling a plant for preventing plague of the present invention. FIG.
Fig. 2 is a process diagram showing a method for producing the composition for controlling plant pandemic of the present invention.
FIG. 3 is a graph showing the growth inhibition effect of the mycelia growth inhibitory effect on the offspring of pepper as a result of the test according to Test Example 1. FIG.
Fig. 4 is a photograph showing the results of the analysis according to Test Example 1. Fig.
Fig. 5 is a photograph showing the results of antibacterial activity of the tomato mold rot fungus according to Test Example 1. Fig.
FIG. 6 is a graph showing the degree of disease occurrence from the first inoculation day to the 7 days after the first inoculation day of the inoculum of pepper rot fungus according to Test Example 3. FIG.
Fig. 7 is a photograph taken at 6 days after inoculation of the composition of Example 1 according to Test Example 3 and the inoculation of pepper plants in the water treatment area.
Fig. 8 is a photograph taken at 10 days after the treatment of tomato mold rot fungus according to Test Example 3. Fig.
9 to 11 show results of organic acid analysis of the medium after harvest of mushroom according to Test Example 4. Fig.
Fig. 12 shows the content of oxalic acid in the extract of Example 1 and the extract of Comparative Example 1 according to Test Example 4. Fig.
Fig. 13 shows the result of examining the degree of antimicrobial activity against pepper blight according to Test Example 5. Fig.
Fig. 14 is a photograph showing the results of photographs showing the antibacterial activity against pepper blight according to Test Example 5. Fig.
Fig. 15 shows the result of examining the degree of antimicrobial activity against tomato wilt disease according to Test Example 5. Fig.
16 is a schematic view showing a plant disease resistance induction mechanism.
Fig. 17 shows the results of qRT-PCR analysis of the pepper blight resistance gene-related gene according to Test Example 6. Fig.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.

The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprises" or "having" and the like are used to specify that there is a feature, a number, a step, an operation, an element, a component or a combination thereof described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Referring to the accompanying drawings, the same or corresponding components are denoted by the same reference numerals, do.

Hereinafter, the composition for controlling plant diseases according to the present invention will be described.

The composition for controlling plant diseases according to the present invention comprises a concentrate of an extract extracted from a medium after cultivation of Lentinula edodes containing oxalic acid as an active ingredient and is used for preventing or controlling plant diseases Can be used.

The plant bottle may be a plant bottle derived from Phytophthora capsici or Ralstonia solanacearum.

Preferably, the extract is an extract by hot water extraction.

The concentration of oxalic acid contained in the concentrate of the extract may be 0.6 to 2.0 mg / ml.

By concentrating the extract, the content of oxalic acid can be increased to improve the antimicrobial activity of the plant disease.

The phytopatholipase-derived plant disease or the plant disease derived from the Ralstonia solanacearum may be a plague against Solanaceae plants, Cucurbitaceae plants, and Fabaceae plants, The composition for controlling plant diseases according to the present invention is mainly effective against pepper blight.

The phytophthora capsaicin-derived plant disease may be pepper blight disease, and the plant disease originating from the Ralstonia solanaceae may be a tomato blight disease.

The plant disease controlling composition may further comprise a preservative.

The preservative is preferably an environmentally friendly preservative, and it is more preferable to apply a preservative that can be used as a food additive.

As a specific example, the preservative may be selected from the group consisting of Potassium Sorbate, Sorbic Acid, Propyl Gallate, Butylated Hydroxy Toluene, Butylated Hydroxy Anisole, .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flowchart sequentially illustrating a method for producing a plant disease controlling composition of the present invention. A method for producing a plant disease controlling composition will be described with reference to FIG.

First, the mushroom is cultivated and the remaining medium is prepared (step a).

The medium remaining after cultivation of the mushroom is preferably the medium remaining after cultivation of mushroom in solid state fermentation.

The solid medium includes sawdust and nutrient source, and the nutrient source may include rice bran, wheat bran, beet pulp, corncob, cotton bark, cotton bark, kraft pulp and the like, but the scope of the present invention is not limited thereto.

The medium remaining after cultivation of the mushroom may be the medium remaining after collecting mushrooms four to eight times in the solid medium. More preferably, the medium remaining after 4 to 6 times of collection can be used.

For example, in the case of a sawdust medium of about 1 kg, when the mushroom development and collection times are three times (90 days) in the culture period of 80 to 100 days, the single cultivation period takes 170 to 190 days. Therefore, the mushroom sawdust medium secretes a large amount of wood-degrading enzymes such as xylanase, cellulase, ligninase and the like during the germline generation in which the mycelial growth and fruiting body are produced, The lignocellulosic fiber is decomposed into a water-soluble low-molecular weight sugar, and the nutrient absorption process of the mycelium can be utilized as an energy source necessary for vital maintenance in vivo.

Therefore, the extraction of antimicrobial active ingredient derived from shiitake mushroom, which is an edible mushroom, and the use of disease control are very useful as an eco-friendly plant disease control material in advance of blocking human harmfulness. The remaining medium after cultivation of shiitake mushrooms may have various characteristics such as a high activity antimicrobial active ingredient in the medium while retaining the characteristics of the solid medium.

Next, the medium is disrupted to prepare a disrupted medium (step b).

The crushing is finely crushed through a crusher and filled into a nonwoven fabric to prepare a raw material.

Thereafter, the shredded medium Heat number  And extracted with oxalic acid ( 산화물 acid ) Is prepared (step c).

The hot water extraction may use a hot water extractor, which is typically used for hot water export as shown. By using such a large-capacity hot water extractor, there is an advantage that a large amount of composition can be produced in a short time.

It is preferable to add the crushed medium to the extraction container of the hot water extractor, add water, and extract hot water for 1 to 3 hours at 70 to 90 ° C to produce a hot water extract.

At this time, the weight ratio of the crushed medium and water to water is preferably 1: 5 to 1:10, more preferably 1: 8 to 1:10.

Next, Heat number  The extract was concentrated to give Heat number Than extract  The content of oxalic acid Increased  A concentrate is prepared (step d).

The concentrate is preferably prepared by concentrating the hot-water extract to 15 to 25-fold, and more preferably 18 to 20-fold.

Accordingly, the antimicrobial activity against the plant disease can be maximized by increasing the content of oxalic acid, and the final content of oxalic acid in the concentrate is increased by at least twice as much as that in the hot-water extract, so that the concentration is 0.6 to 2.0 mg / ml Do.

The preparation of the concentrate can reduce the storage volume and can be diluted to an appropriate concentration at the time of use.

Finally, the preservative is added to the concentrate (step e).

Since the kind of the preservative is the same as that described in the above-described composition for controlling plant diseases, the details of the preservative will be referred to.

[Example]

Example  One

2 is a process diagram showing the process of the first embodiment of the present invention. Hereinafter, the first embodiment will be described with reference to FIG.

A medium comprising 80% of sawdust and 20% of rice bran are prepared, the medium is sterilized by autoclaving for 2 hours or more and cooled, then inoculated with the seed bacterium, the mycelial growth nutrition reproduction step and the medium change step of 60 days or more, . After shrimp fruiting body growth and growth stage, the whole was harvested for about 90 days and harvested 4 to 8 times.

50 kg of the thus-prepared shiitake mushroom-harvested medium was finely crushed in a crusher to prepare a crushed medium and filled into a nonwoven fabric. Then, the crushed medium was placed in an extraction container in the large capacity hot water extractor as shown, 450 L of water was added, and the hot water extract was prepared by hot water extraction at 80? For 2 hours. Next, the hot water extract was concentrated 20 times to prepare a concentrate of the composition for controlling plant diseases, which is a liquid formulation of 50 L. Finally, 0.2% antiseptic potassium salt, which is used as an organic material, was added to the plant disease control composition to prevent decay.

Comparative Example  One

The composition concentrate was prepared in the same manner as in Example 1, except that the shiitake medium, that is, the shiitake medium without shiitake mushroom was used instead of the medium after shiitake cultivation.

[Test Example]

Test Example  1: After shiitake mushroom harvest Of hot-water extract  Antimicrobial effect

The concentrate prepared according to Example 1 and Comparative Example 1 was diluted 10-fold each to prepare a diluted solution. 3.9 g of powdered potato dextrose agar (PDA) powder was added to 100 ml of the composition of each of diluted Example 1 and Comparative Example 1, sterilized at 121 ° C for 20 minutes, and dispensed into a Petri dish.

The mycelial growth rate was examined from day 1 to day 7 after inoculation of a pre-grown mycelium slice having a diameter of 5 mm in the center of the petri dish. As a result, a graph showing changes in mycelial diameter with the elapsed days after the inoculation of mycelium is shown in Fig. 3, and the growth of the mycelium at 7 days is shown in Fig.

3 and 4, it was confirmed that the composition of Example 1 suppressed the growth of mycelium of P. perilla in more than 60% from the initial stage of inoculation, as compared with the composition of Comparative Example 1. In addition, when 7 days passed, the mycelium of P. aeruginosa grew by 9 cm in the petri dish treated with the composition of Comparative Example 1, whereas the petri dish treated with the composition of Example 1 had a diameter of 3.5 cm and a mycelial growth of 62% .

The antimicrobial activity of the plant pathogenic bacterium, Tomato spp. (Ralstonia solanacea), was determined using a paper disk assay, and the results are shown in FIG. Specifically, the base medium was a NA (nutrient agar) medium, and 2 x 10 ⁶ of a cell suspension of Tomato phytophthora infestans was added to NB (Nutrient Broth) containing agar of 0.8% as a smear medium.

Then, 70% methyl alcohol extract (disc 2), hot water extract (disc 3) and hot water extract (disc 4) of the medium after harvesting were added to the medium, And the amount of antimicrobial activity was measured after 3 days by the size of clear zone formed around the paper disk. At this time, the above-mentioned hot water and hot water extracts of Disc 1 and 2 were extracted at room temperature for one hour and the hot water extracts of Disc 3 and 4 were extracted at 80 ° C for 1 hour, respectively.

According to FIG. 5, the clear water hardly formed in the hot water extract (disc no. 2) of the original medium and the 70% methyl alcohol extract (disc no. 3) of the post-harvest medium, And the hot water extract (disc no. 3) of the post-harvest medium showed a clear clear zone of 20 mm or more. Thus, it was confirmed that the antimicrobial effect was good.

Therefore, it was confirmed that the composition prepared according to Example 1 of the present invention has antimicrobial activity in pepper blight caused by fungus germs and tomato fungus, a bacterium.

Test Example  2: Analysis of promotion effect of pepper growth

The composition of Example 1, in which the pepper seeds were seeded in a 40-well pot containing the horticultural soil and grown to seedlings for 3 weeks, and then transplanted to a horticultural soil contained in a pot (10 cm in diameter) After 20 days, the fresh weight, leaf length, leaf width, leaf number, and biomass of root and root were measured. The results are shown in Table 1 below.

According to Table 1, it can be seen that the composition of Example 1 was increased by 40% of the plant length, 42% of the leaf number, 70% of the ground biomass, and 50% of the root biomass compared to the water treatment. , 24% of plant height, 14% of leaf number, 58% of ground biomass, and 50% of root biomass.

These results indicate that the composition prepared according to Example 1 of the present invention contains not only the control of pepper blight, but also a useful ingredient for promoting pepper growth. In the mushroom culture medium, mushroom mycelium secretes a large amount of woody degrading enzymes such as xylanase, cellulase and ligninase, and absorbs glucose, which is a decomposition product of wood fiber, and cultivates it in the process of using it as an energy source It is considered that a large amount of decomposition products remain in the post-culture medium, and various nutrient sources produced by mushroom mycelium such as amino acid and nitrogen are contained, and thus it can be used as a carbon source and nitrogen source necessary for plant growth.

Test Example  3: Pepper Plague  Effect analysis of tomato blight disease control

In order to induce disease in the pepper plants, the host was produced and used as an inoculum for the development of the pest in the pepper plants. To produce the red pepper platelets, a small pepper reverse mycorrhizal flora (5x5 mm) was inoculated at the same distance to five sites on the medium containing 10% V8 juice purchased from the market and containing 2.0% Agar. This mycelial slice was placed on a Petri dish, and sterilized water (20 ml) was added thereto, followed by incubation at a temperature of 24 ° C for 3 days under fluorescent light irradiation . At this time, the zoosporangia on the mycelial section was confirmed by high power (X400) optical microscope (Zeiss Axio imager). 4? The mixture was placed in a refrigerator for 60 minutes and then allowed to stand at room temperature for 30 minutes. The outflow was observed in a high magnification (X400) optical microscope and the population was adjusted to a concentration of 10 ⁵ ml ^-1 using a hematocytometer. Respectively.

 The red pepper plants were cultivated in 40 days after sowing, using "Bukwang", a sensitive cultivar for red pepper.

In the test method, 50 ml of each of the diluted solution and water was diluted 10 times with respect to the control composition of Example 1, and 50 ml of water was inoculated into each of the capsules of the pepper seedlings. After 3 days, 1 ml of the pepper sterilized inoculum was inoculated on the root side of the pepper plant, Were observed.

The degree of disease occurrence was observed from the first inoculation day to the 7 days after inoculation of the pepper rot fungi inoculum and is shown in FIG. In this case, the degree of disease occurrence is a relative value of 1 to 5 (1: 5-10% leaves begin to vigorously, 2: 20-30% plant wilt, 3: 40-50% 70% plant wilting, 5: total plant wilting and grayish brown stalks at base).

The control rate was calculated by the following equation (1).

[Equation 1]

Control rate (%) = 100 (1-x / y)

Here, x is the treatment group and y is the non-treatment group.

 According to FIG. 6, the degree of onset was 2 in water treatment from 2 days after inoculation with pepper rot, and the degree of onset was 0.35 in the composition of Example 1, showing a control ratio of 82% or more. As the time passed, the disease progressed even in the composition of Example 1, and the control rate was reduced by about 50% compared to the water treatment after 8 days of inoculation.

Fig. 7 is a photograph taken at 6 days after inoculation of pepper rot fungi in the composition treatment and water treatment in Example 1. Fig. According to Fig. 7, it can be confirmed that the composition of Example 1 is growing healthily, while the water treatment area shows the inherent symptom of P. aeruginosa seeded by whole plants.

Fig. 8 shows the control effect against tomato blight disease, which was photographed at 10 days after treatment with tomato blight. According to FIG. 8, it was confirmed that the tomato plants were wilted in water treatment but the composition treatment of Example 1 was alive and well.

Test Example  4: Analysis of organic acids in medium after shiitake mushroom harvesting

The mycelium of shiitake mushroom was inoculated into 1000 ml of Potato Dextrose Broth (PDB) liquid medium, cultured at 25 ° C for 30 days with shaking, and further cultured for 20 days. As a result of measurement with a pH meter, it was found that the pH was 3.9, which was an acidic solution. Organic acid analysis was carried out and the results are shown in FIGS. 9 to 11.

HPLC analysis conditions for the organic acid analysis were Aminex HPX-87 column 4.6 (ID) x 300 mm, flow rate 0.6 ml / min, 4 mM H ₂ SO ₄ , and detector UV 215 nm. As shown in FIG. 9, the organic acid analysis showed that phytic acid, oxalic acid, malic acid, malonic acid, lactic acid, formic acid, fumaric acid ), Butyric acid, and isobutylic acid were detected.

In order to quantify oxalic acid, oxalic acid standard product (Sigma, purity 99%) was purchased to make a calculation line for various concentrations of oxalic acid (200, 500, 1000, 2000 ppm) And the correlation coefficient (R ² ) of oxalic acid was confirmed through a calculation line prepared according to FIG. According to FIG. 10, it can be confirmed that the linearity (R ² > 0.9982, which indicates accuracy of 99.82%) is confirmed.

In addition, the concentration of oxalic acid contained in the mushroom culture (1 L) is shown in Fig. According to Fig. 11, the concentration of oxalic acid contained in the cultured shiitake medium (1 L) was 708.711 ppm. In addition, oxalic acid content was calculated by the following formula 1 and expressed as 0.07%. In other words, it was found that oxalate of 0.7 mg / ㎖ was contained in the mushroom culture.

[Equation 1]

On the other hand, FIG. 12 shows the results of comparing oxalic acid contents in the composition concentrate of Example 1 and the composition concentrate of Comparative Example 1. According to Fig. 12, the phytic acid content was overlapped in Example 1 and Comparative Example 1. However, in Example 1, a single peak at 6.62 minutes indicating oxalic acid was detected, and 1.5 mg / ml of oxalic acid Which is more than two times. That is, the present invention is characterized by enhancing antimicrobial activity by increasing the content of oxalic acid by concentration of the plant disease controlling composition of the present invention. On the other hand, oxalic acid was not detected in Comparative Example 1.

Test Example  5: Antibacterial effect analysis

First, the degree of antimicrobial activity against the pepper blight of PDA (potato dextrose agar) medium containing the concentrate prepared according to Example 1 and oxalic acid was examined, and the results are shown in FIG. 13 and FIG. Specifically, the mycelial growth of pepper blight was investigated 7 days after inoculation of mycelia of pepper blight into PDA (Potato dextrose agar) medium containing oxalic acid (Sigma) at final concentrations of 0, 100, 150, 250, 300 and 400 ppm . 13 and 14, it was confirmed that the mycelial growth of the pepper blight was inhibited by 22% to 55% by the oxalic acid treatment concentration 7 days after the inoculation, and the concentrate prepared according to Example 1 had mycelial growth of up to 70% And the antimicrobial activity against pepper blight was remarkably high.

Next, the degree of antimicrobial activity against tomato blight disease according to the concentration of oxalic acid was examined, and the results are shown in FIG. Specifically, in order to investigate the inhibitory effect on tomato oxalic acid bacteria on oxalic acid, the concentration of oxalic acid in 5 ml of Nutrient liquid medium was changed from 5000 ppm to 100 ppm to 5 × 10 ³ for the first time for 18 hours As a result, the growth of bacteria was inhibited when 250-5000 ppm of oxalic acid was contained. However, when the content of oxalic acid was lower than that of bacteria, the growth of bacteria was similar to that of the control water treatment.

Test Example  6: Plant disease  Resistance induction effect test

The plant disease resistance induction mechanism is schematically shown in Fig. 16, a disease-resistant derivative (elicitor) is recognized in a receptor in host cells and stimulates disease-resistance inducing signal transduction substances such as salicylic acid (SA) or jasmonic acid (JA) Lt; / RTI > Induction resistance has systemic acquired resistance (SAR) and induced systemic resistance (ISR), and SAR is SA dependent pathogenesis-related (PR) gene including PR-1a and PR-4 And ISRs are known to induce other PR genes, such as plant defensin 1.2, as a signal transduction system dependent on acid and ethylene (ET).

The relationship between the composition concentrate of Example 1 and the pepper blight resistance response was examined. Specifically, 50 ml of the composition of Example 1 was treated with pepper seedlings and the total RNA was isolated after 96 hours. Using the primers capable of amplifying the pepper resistance gene of Table 2, qRT- PCR analysis was carried out and the results are shown in Fig.

Pepper resistance gene Primer base sequence CaPR1 F: 5'-ACTTGCAATTATGATCCACC-3 '
R: 5'-ACTCCAGTTACTGCACCATT-3 '
CaBGLU F: 5'-TAAAAGGGGAAGTCCAAGAAGG-3 '
R: 5'-TCAGCAAAAATGTCCAAAAATC-3 '
CaPR4 F: 5'-AACTGGGATTTGAGAACTGCCAGC-3 '
R: 5'-ATCCAAGGTACATATAGAGCTTCC-3 '
CaPR10, F: 5'-ATGTTGAAGGTGATGGTGGTGCTG-3 '
R: 5'-TCCCTTAGAAGAACTGATACAACC-3 '
CaActin F: 5'-TTGGACTCTGGTGATGGTGTG-3 '
R: 5'-AACATGGTTGAGCCACCACTG-3 '

PCR reaction mixture (2 X SYBR Green 1 master mixtures (Roche), cDNA, 10 pmol qRT-PCR primer) was used to make the final 20 μl. The above reaction composition was subjected to PCR amplification conditions at 95 ° C. for 15 minutes, 58 ° C. for 30 seconds, 72 ° C. for 30 seconds in a LightCycler 96 system (Roche, USA). Gene expression quantification was performed using a software program .

17, the expression levels of the resistance genes CaPR1, CaPR4, CaBGU, and CaPR10 in the Example 1 treatment (WESMS) were increased from 4 times to 15 times that of the water treatment treatment. The CaPR1, CaPR4 and CaPR10 resistance genes induced resistance gene expression higher than that of the commercial DL-β-aminobutyric acid (BABA, 0.5%), which is known as a resistance gene expression derivative. This indicates that the composition of Example 1 expresses a disease-resistant gene.

CaPR1 and CaPR4 show salicylic acid-dependent systemic resistance, in which salicylic acid can accumulate in plants. Thus, when the composition of Example 1 was treated, the increase in the content of salicylic acid in the pepper plants was analyzed by a liquid chromatograph mass spectrometer (LCMS8050), and the results are shown in Table 3 below.

Treatment Salicylic acid content (ng / ml) 48 hours 96 hours 120 hours Example 1 12.77 ± 1.34 12.79 ± 1.65 18.68 + - 0.41 DL-β-aminobutyric acid (BABA, 0.5%) 26.06 ± 1.62 31.62 ± 1.92 14.85 ± 1.90 Water treatment 5.24 ± 1.27 3.49 ± 0.71 5.97 ± 1.73

According to Table 3, the salicylic acid content was increased about 2.5 times from the 5.24 mg / ml of the water treatment from 48 hours after the treatment of the composition of Example 1 to 12.7 mg / ml, and increased more than 3 times after 120 hours appear. Therefore, it is judged that the composition of Example 1 is treated with a pepper plant to function as a derivative for inducing disease resistance gene expression by producing salicylic acid.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, many modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims. The present invention can be variously modified and changed by those skilled in the art, and it is also within the scope of the present invention.

Claims (17)

A method for preventing or controlling plant diseases, which comprises concentrating the extract obtained from the medium after cultivation of Lentinula edodes containing oxalic acid as an active ingredient,
The botanical bottle is a plant bottle derived from phytoplasma capsaicin or a plant bottle derived from Ralstonia solanaceae,
Wherein the concentration of oxalic acid contained in the concentrate of the extract is 250 to 5000 ppm. The method according to claim 1,
Wherein the extract is an extract from hot water extraction. delete delete delete delete The method according to claim 1,
Wherein the composition for controlling plant diseases further comprises a preservative. 8. The method of claim 7,
The preservative may be selected from the group consisting of Potassium Sorbate, Sorbic Acid, Propyl Gallate, Butylated Hydroxy Toluene and Butylated Hydroxy Anisole Wherein the plant disease prevention composition is one of the following. a) preparing the remaining medium after cultivating shiitake mushroom;
b) disrupting said medium to produce a disrupted medium;
c) hydrothermally extracting the shredded medium to produce a hot-water extract containing oxalic acid; And
d) concentrating the hot-water extract to produce a concentrate having an increased content of oxalic acid than the hot-water extract,
In the step c), the crushed medium is added to the hot water extractor, water is added, and the hot water extract is prepared by hot water extraction at 70 to 90 ° C for 1 to 3 hours,
The step d) is to concentrate the concentrate so that the oxalic acid content of the concentrate is 250 to 5000 ppm to prepare a composition for controlling plant diseases,
Wherein the plant disease caused by phytophthora capricidis is pepper blight or the plant disease derived from Ralstonia susanaceae is a tomato blight disease. delete 10. The method of claim 9,
After step d)
e) adding a preservative to the concentrate. < RTI ID = 0.0 > 8. < / RTI > delete delete 10. The method of claim 9,
Wherein the weight ratio of the crushed medium to water in step c) is 1: 5 to 1:10. 10. The method of claim 9,
Wherein the remaining medium after cultivation of the mushroom is the medium remaining after cultivation of mushroom in a solid state fermentation. 16. The method of claim 15,
The solid medium comprises sawdust and a nutrient source,
Wherein the nutrient source comprises at least one selected from the group consisting of rice bran, wheat bran, beet pulp, corncob, cotton seed bark, cotton bark, and kraft bark. 16. The method of claim 15,
Wherein the remaining medium after cultivating the mushroom is the medium remaining after collecting mushroom four to eight times in the solid medium.

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