KR20190086945A - Hypoxylon perforatum KDC0114 strain having antifungal activity against pathogens and plant growth promoting effect, and uses thereof - Google Patents

Abstract

The present invention relates to a Hypoxylon perforatum KDC0114 strain having a plant disease control or plant growth promoting effect and uses thereof. The strain has the effect of controlling soft rot of tobacco or Arabidopsis thaliana, and has the effect of promoting the growth of plants. In addition, volatile organic substances secreted by the strain have the effect of promoting growth and controlling the soft rot of tobacco or Arabidopsis thaliana. Therefore, the Hypoxylon perforatum KDC0114 strain of the present invention can be usefully used as an environmentally friendly microbial agent such as a biocontrol agent and plant growth promoter.

Description

Hypoxylon perforatum KDC0114 strain having a plant disease control or plant growth promoting effect and its use (Hypoxylon perforatum KDC0114 strain having antifungal activity against pathogens and plant growth promoting effect, and uses thereof)

The present invention relates to a method for controlling a plant disease or a plant growth promoting effect of Hypoxylon perforatum KDC0114 strain, a culture thereof, and a method for treating them.

In order to control the plant diseases caused by plants in order to increase the production amount in agriculture in general, pesticides based on organic compounds have been manufactured and used all over the world. Recently, there has been a serious problem of contamination of agricultural products with the problem of environmental pollution. Therefore, many agricultural fertilizers using high quality organic fertilizers have been developed rather than chemical fertilizers, and many organic fertilizers have been developed and used As an alternative to chemical fertilizers, organic fertilizers that are used extensively differ only in their material properties, and they are exacerbating other problems similar to chemical agents such as soil contamination, chain failure, imbalance of soil nutrients, and the occurrence of pests.

As a means to replace the chemical fertilizer in order to solve the above problems, biological methods for solving safe food while minimizing agricultural environment and ecosystem burden are being actively researched. As an alternative method, researches on disease control using useful microorganisms Is constantly being done. The main microorganisms used zero biocontrol is Bacillus (Bacillus) (Bacillus bell Debrecen cis NS05 strain and Korea registered microbial agent for controlling plant diseases, comprising the same Patent No. 0817428 No.), Pseudomonas species (Pseudomonas), genus Streptomyces ( Streptomyces ) and Enterobacter , and fungi such as Ampelomyces and Trichoderma are known.

The microorganisms used in these studies are microorganisms harmless to the environment and the human body. The microorganism fertilizer using the microorganisms has an advantage of not causing environmental problems of soil pollution such as salt accumulation, and is excellent in maintaining the stability of the ecosystem and continuously improving crop productivity There is an advantage.

However, despite the fact that the development technology of microbial agents is improving in Korea, the level of development is still insignificant compared to the advanced countries, and microbial agents consumed in the domestic market tend to be imported from abroad.

Meanwhile, Hypo Ron Giles (Hypoxylon) genus ascus typically found in a dead tree fungi (Ascomycetes) Flam. However, recent studies have also shown that the fungi are the endogenous fungi of living trees. Some species belonging to the genus Hypogylon have been identified as one of the major medicinal and cooking fungi in China and Taiwan, fuciformis ) can be used for the cultivation of white mushroom (white mushroom fungus, high- density cyanuric lujibinosus ASI 6046 strain, and artificial cultivation method of white chestnut mushroom using the same, 0252384), a microbial agent for controlling plant diseases or promoting plant growth using a strain of the genus Hyponylon has not been disclosed.

Accordingly, the present inventors have succeeded in isolating a microorganism belonging to the genus Haemophilus, and confirming that the isolated microorganism has a plant disease control or plant growth promoting effect, so that the microorganism can be used as a microbial agent for plant disease control or plant growth promotion .

The inventors of the present invention have made efforts to develop a microbial agent for controlling environment-friendly plant diseases or promoting plant growth that can solve the problems of conventional organic or chemical fertilizers. As a result, the inventors of the present invention have found that Hypoxylon perforatum ) KDC0114 strain has a plant disease control effect or a plant growth promoting effect, thus completing the present invention.

Accordingly, the present invention relates to a method for controlling plant diseases or promoting plant growth, which comprises administering a composition comprising Hypoxylon perforatum ) KDC0114 strain and a microorganism preparation using the same.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention as described above, the present invention provides a method for controlling plant diseases or promoting the growth of plants by using Hypoxylon perforatum ) strain KDC0114 (KCTC 13442BP).

In one embodiment of the present invention, the strain may secrete a volatile organic substance having a plant disease control effect or a plant growth promoting effect.

In another embodiment of the present invention, the botanical bottle may be a cigarette or a pyramidal bottle of Arabidopsis.

In addition, the present invention relates to the aforementioned Hypoxylon < RTI ID = 0.0 > perforatum KDC0114 strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof as an active ingredient. .

The present invention also provides a microbial fertilizer for controlling plant diseases, comprising the microbial agent.

The present invention also provides a method of controlling plant diseases, comprising the step of treating the microorganism preparation with a soil, a plant, or a seed of a plant.

In addition, the present invention relates to the aforementioned Hypoxylon < RTI ID = 0.0 > perforatum ) KDC0114 strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof, as an active ingredient, to a plant growth promoting microorganism .

In addition, the present invention provides a microorganism fertilizer for promoting plant growth, comprising the microorganism preparation.

The present invention also provides a plant growth promoting method comprising the step of treating the microorganism preparation with a soil, a plant, or a seed of a plant.

Hypoxylon according to the present invention perforatum ) The strain KDC0114 has the effect of controlling cigarette or scab disease of Arabidopsis and has the effect of promoting plant growth. In addition, it has been confirmed that the volatile organic compounds secreted by the strain have an effect of controlling the cicatric disease of the tobacco or Arabidopsis and accelerating the growth of the cigarette or the Arabidopsis thaliana. As a result, it was confirmed that Hypoxylon perforatum ) KDC0114 is expected to be useful as an environmentally friendly biocontrol agent and plant growth promoter.

Figure 1 is a graphical representation of the results of a < RTI ID = 0.0 > Hypoxylon < / RTI > perforatum) KDC0114 strain of the present invention.
FIG. 2 is a graph showing the results of a test for the control of acanthosis of Arabidopsis thaliana by Hypoxylon perforatum strain KDC0114 according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of a test for promoting the growth of tobacco by a volatile organic substance of Hypoxylon perforatum strain KDC0114 according to an embodiment of the present invention.
FIG. 4 is a graph showing the results of a test for the control of tobacco rash disease caused by a volatile organic substance of Hypoxylon perforatum strain KDC0114 according to an embodiment of the present invention.
5 is a diagram showing the ITS region base sequence of Hypoxylon perforatum KDC0114 strain according to an embodiment of the present invention.

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.

Hereinafter, the present invention will be described in detail.

The object of the present invention is to provide Hypoxylon perforatum KDC0114 strain (KCTC 13442BP) having plant disease control or plant growth promoting effect.

The ITS region sequence of the strain was 99% homologous to Hypoxylon perforatum and was found to be a novel strain with 1% homology between the strains.

Thus, the present inventor named the novel strain obtained as Hypoxylon perforatum KDC0114 strain and deposited it on KCTC (Korea Research Institute of Bioscience and Biotechnology) on Jan. 2, 2018, 13442BP.

The strain of the present invention can be obtained by isolating and identifying from the rhizosphere of the soil harvesting obtained from the Dokdo Island map, and has a plant disease control or plant growth promoting effect.

The botanical bottle may be a cigarette or a scurf bottle of Arabidopsis.

The Hypoxylon < RTI ID = 0.0 > (see FIG. 1 and Experimental Example 1) in the cigarette treated with the perforatum KDC0114 strain, it was confirmed that even in the Arabidopsis thaliana treated with the strain, a higher control effect of the scurf disease was obtained compared with the negative control (See Fig. 2 and Experimental Example 2).

At this time, the cocaine of the cigarette is caused by the pathogen Pectobacterium carotovorum subsp. Carotovorum (or Erwinia carotovora subsp. Carotovora ) . The pathogen belongs to the pathogenic organisms and has a size of 1.5 to 3.0 × 0.6 to 0.9 μm and 2 to 10 hairs. The optimal temperature for growth is 32 ~ 35 ℃, maximum 40 ℃, minimum 0 ~ 9 ℃, and the host range is very wide, damaging the branches, flora, asteraceae, lilies and butterflies. In addition, the pathogens can survive in the soil for a long period of time, propagate during the irrigation of the bulb, infiltrate through the wound on the plant, and occur when the temperature is high and the temperature is high and the condition is dry.

Also, tobacco rash disease occurs by the pathogen in leaf stalk, pedicel and bulb of tobacco, and part of leaf stalk is formed by corolla corruption, and it gradually expands to soften leaf and stem. If the cigarette scarlet disease is severe, the whole leaf becomes yellow, and eventually it will die. The bulb of the cigarette which is caught in the cigarette scarlet disease rotates in black brown color and the upper part dies and dies.

On the other hand, the Hypoxylon of the present invention it was confirmed that VOCs (Volatile Arganic Compounds) secreted by perforatum KDC0114 promotes the growth of plants and exhibits a plant disease control effect (see FIGS. 3, 4, and 3).

Accordingly, the present invention relates to a method for producing a poly perforatum ) KDC0114 strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof, as an active ingredient. A microorganism preparation for accelerating plant growth and a fertilizer can be provided. The microorganism preparation and the fertilizer are treated to provide a plant disease control method and a plant growth promotion method.

The microorganism preparation of the present invention can be used in tobacco, Arabidopsis, etc., and the preparation can be used without being limited to the kind of the plant.

In the present invention, the culture is preferably obtained from a PDA (Potato dextrose agar) medium or a V8A (V8 juice agar) medium in which the strain has been cultured, but is not limited thereto.

The microbial composition may be formulated for controlling plant diseases and promoting plant growth by a conventional method, and may be prepared in the form of a dry powder or a liquid fertilizer. Specifically, the microbial drug composition according to the present invention may be prepared in the form of a liquid fertilizer, and the diluent may be added to the microbial pharmaceutical composition in the form of a powdered powder, or may be granulated by formulating it. However, the formulation is not particularly limited. Preferably, it can be formulated as a plant growth-promoting biologic fertilizer for replacing chemical fertilizer, that is, it can be formulated as a biological fertilizer to overcome it in environment-friendly organic agriculture with limited supply of chemical fertilizer.

In the present invention, the microorganism preparation can be prepared by adding an additive such as a surfactant, a weight agent, and a nutrient to a strain or a culture thereof. The surfactant may be selected from the group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Polyoxyethylene alkylarylpolyether, polyoxyethylene nonylphenyl ether, sodium sulphonate naphthalene formaldehyde, Triton 100 and Tween 80. The composition of claim 1, And one or more selected from the group consisting of soybean flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose, and starch may be used as the extender and the nutrient. Examples of the disintegrant include bentonite bentonite, talc, dialite, Kaolin and calcium carbonate may be used in the present invention.

The present invention also provides a method for controlling plant diseases and a method for promoting plant growth by treating the microorganism preparation with a soil, a plant or a plant seed. At this time, the treatment methods include a general method (for example, spraying, misting, atomization, powder spraying, granule spraying, water surface application, constant use), soil application (for example, mixing, , Surface use (for example, coating, smearing, coating, etc.), immersion, poisoning, smoke application, and the like. The amount of usage can be appropriately determined according to the formulation, the damage situation, the application method, the application site, and the like.

In the present invention, the effective amount of the microorganism contained in the preparation to be treated according to the above method can be included in the number of microorganisms of 1 to 1 x 10 < 1 > per cropland area (m & In addition, an effective amount of the microorganism contained in the formulation to be treated by spraying among the above methods can be included at a microbial concentration of 1 to 1 x 10 < [chi] > per ml and the effective amount of the microorganism contained in the composition to be treated by immersion is 1 To 1 x 10 < RTI ID = 0.0 > ¹⁰⁰ < / RTI >

Hereinafter, preferred embodiments and experimental examples of the present invention will be described in detail with reference to the accompanying drawings. However, these examples and experimental examples are for illustrating the present invention only, and the scope of the present invention shall not be construed as being limited by these examples and experimental examples.

Example 1 . Inductive resistance - Defense mechanism  And fungi that induce plant growth Hypo xylone  Isolation, identification and cultivation of perl formazan KDC0114

Hypoxylon according to the present invention perforatum ) KDC0114 strain was deposited on Jan. 02, 2018 with the Genetic Resource Center in the Biotechnology Research Institute of Korea Research Institute of Science and Technology (KCTC 13442BP).

As for the process of isolating the fungi, the ground cutting slices and the soil attached to the roots collected from Dokdo Island (north latitude: 37.14.330, longitude: 131.52.133) were shaken with sterilized distilled water, And transferred to a nutrient medium, Potato dextrose solid medium (PDA). At this time, 25 μg / mL chloramphenicol was added to the solid medium to inhibit the growth of bacteria. The culture of the fungi was incubated at 25 ° C for 5 days, and this process was repeated several times until the isolated fungi were isolated.

In order to identify the isolate, the nucleotide sequence of the internal transcribed spacer region (ITS) of the strain was obtained. In order to investigate the homology of the previously registered fungi with the ITS region sequences, the following data were collected from Unite (https://unite.ut.ee/) and NCBI (https://www.ncbi.nlm.nih.gov/) was used to provide service to blast, so that the hypo-xylene Ron Pearl Fora Terme (Hypoxylon perforatum) and was found to have a 99% homology, this hypo-xylene Ron Pearl Fora Terme (Hypoxylon perforatum ) KDC0114.

The fungus of the present invention, Hypoxylon < RTI ID = 0.0 > perforatum ) The nucleotide sequence of the ITS region of KDC0114 is shown in Fig. The strain has the PDA and V8A (V8 juice agar) medium as the optimum medium, and has the growth temperature of 25 占 폚.

Experimental Example 1 . Hypo xylone Pearl Forlator KDC0114  Of cigarette by strain treatment Sickle disease  Control effect

Tobacco seeds were sterilized with 1.2% sodium hypochlorite for 15 minutes, rinsed three times with sterilized water for 10 minutes, seeds were sprayed on MS (Murashige-Skoog) medium and germinated at 25 ° C for 3 days. The germinated tobacco seeds were transferred to a 24-well culture plate containing 1.2 ml of MS medium and incubated at 25 ° C for 2 weeks. Hypoxylon pterorum KDC0114 fungi isolated in Example 1 was cultured in V8A medium for 5 days, and the medium and mycelium were cut to a size of about 2 mm x 2 mm using a flame-sterilized surgical blade, After sealing, the cells were incubated at 25 ℃ for 14 hrs / 10 hrs. For 5 days until the mycelium of the fungus penetrated into the plant roots. Sterile saline was used as a negative control in this experiment. In order to verify the induction resistance-defense mechanism of the strain, 10 ⁸ cfu / ml of Pectobacterium carotovorum subsp. carotovorum suspension was dropped on the leaves of the first branch by 5 μl, and the leaf number was examined by observing for 3 days after pathogen treatment. As a result, as shown in Fig. 1, tobacco treated with hypoxanthone perforatum KDC0114 strain showed higher resistance to the disease than the sterilized saline negative control.

Experimental Example 2 . Hypo xylone Pearl Forlator KDC0114  Effect of Strain Treatment on Arabidopsis thaliana Col-1

Plant preparation of this experiment, inoculation of Hypo xylon pearl poratorum KDC0114 strain and Pectobacterium carotovorum subsp. Carotovorum was treated in the same manner as in Experimental Example 1 above. As a result, as shown in FIG. 2, it was confirmed that the Arabidopsis thaliana treated with the Hyposylon Pearl Poratorum KDC0114 strain was more effective than the sterilized distilled water as the negative control group.

Experimental Example 3 . Hypo xylone Pearl Forlator KDC0114  Promotion of Plant Growth and Disease Control by Secretion of Volatile Organic Substances

Tobacco seeds were sterilized with 1.2% sodium hypochlorite for 15 minutes, rinsed three times with sterilized water for 10 minutes, seeds were sprayed on MS (Murashige-Skoog) medium and germinated at 25 ° C for 3 days. The I-plate is a plate made of two compartments and made to move only between the gases without direct interchange. For the experiment, MS medium was dispensed in both chambers, and 2 mm x 2 mm hyphae of hyphae xylon perforated KDC0114 fungi isolated in Example 1 were contained on the other side of the cigarette germinated in one cell Two agar blocks were incubated at 25 ° C for 14 hours and 10 hours for dark. As a result, after 5 days, there was a noticeable difference in the growth of plants, and the difference became evident over time, and the result is shown in FIG. It has been confirmed that the fungicidal volatile organic material promotes the growth of plants.

To confirm whether the fungus-producing volatile organic compounds are related to the defense mechanism of the plant, the same tobacco and the hypoxanthone peroxidase KDC0114 fungus were inoculated on the I-plate and cultured. One week later, 2 μl of 10 ⁸ cfu / ml cigarette scar suspension was dropped onto the two leaves of the first branch, and the symptoms were observed 3 days later. As a result, as shown in Fig. 4, it was confirmed that the disease occurrence was remarkably reduced in the plants exposed to the volatile organic substances of Hypo xylon pearl porcum KDC0114.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC13442BP

Checked on: 20180102

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Hypoxylon perforatum KDC0114 strain having antifungal activity          against pathogens and plant growth promoting effects, and uses          the <130> MP17-305 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 610 <212> DNA <213> Hypoxylon perforatum KDC0114 ITS region <400> 1 tgcggaggga tcattactga gttctacaaa actcccaacc ctttgtgaat tataccttag 60 ttgcctcggc gtcgagcgag tcaacctacc cgggagctac actggagtca cctaccctgt 120 agaacctacc ctgtaggacc taccctgtag acggctaccc tggagctacc ctgtagttgc 180 actttcgcgt cgccggtgga ctaccaaact cttatatgta tagtgtatct ctgaattctt 240 aacaaaatta gttaaaactt tcaacaacgg atctcttggt tctggcatcg atgaagaacg 300 cagcgaaatg cgataagtaa tgtgaattgc agaattcagt gaatcatcga atctttgaac 360 gcatattgcg cccagtagta ttctactggg catgcctatt cgagcgtcat ttcaaccctt 420 acgccctgta gcgtagtgtt aggactctac tctttagaga gcagttccct aaaaccagtg 480 gcagtgttct gtacactcat agcgtagtaa ttcttctcgc ttctgcagtg gcccgtacta 540 ttcgccgtaa aaccccctat tttctaatgg ttgacctcgg attaggtagg aatacccgct 600 gaacttaagc 610

Claims (9)

Hypoxylon perforatum strain KDC0114 (KCTC 13442BP) having plant disease control or plant growth promoting effect.
The method according to claim 1,
Characterized in that the strain secretes volatile organic substances having a plant disease control or plant growth promoting effect. The Hypoxylon &lt; RTI ID = 0.0 &gt; perforatum ) KDC0114 strain.
The method according to claim 1,
The plant diseases which are characterized by sores tobacco or Arabidopsis etiology, Hypo Ron Giles Pearl Fora Terme (Hypoxylon perforatum ) KDC0114 strain.
A method for producing a strain of Hypoxylon perforatum KDC0114 according to any one of claims 1 to 3, a culture of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof Wherein the microbial agent is a microbial agent for controlling plant diseases.
A microbial fertilizer for controlling plant diseases, comprising the microbial agent of claim 4.
A method for controlling plant diseases, comprising the step of treating the microbial agent of claim 4 to a soil, a plant, or a seed of a plant.
A method for producing a strain of Hypoxylon perforatum KDC0114 according to any one of claims 1 to 3, a culture of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof Wherein the microorganism is one or more microorganisms.
A microbial fertilizer for promoting plant growth, comprising the microbial formulation of claim 7.
A method for promoting plant growth, comprising the step of treating the microbial agent of claim 7 to a soil, plant, or plant seed.

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