KR20190086947A - Erwinia gerundensis KUDC9201 strain having antifungal activity against pathogens, and uses thereof - Google Patents

Abstract

The present invention relates to an Erwinia gerundensis KUDC9201 strain having a plant disease control effect and uses thereof. The strain has the effect of controlling the soft rot of tobacco leaves and bacterial spot disease of pepper, and the strain has been confirmed to control the bacterial spot disease of pepper by increasing the expression level of CaPR4 or CaChi2, the defense genes of pepper. Therefore, the Erwinia gerundensis KUDC9201 strain of the present invention may be usefully used as an environmentally friendly microbial agent such as a biological control agent to activate the plant′s defense genes, thereby enhancing the plant immunity.

Description

Erwinia gerundensis KUDC9201 strain having plant disease control effect and its use {Erwinia gerundensis KUDC9201 strain having antifungal activity against pathogens, and uses thereof}

The present invention relates to an Erwinia gerundensis strain KUDC9201 having a plant disease control effect, a culture thereof, and a method of 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 improved in Korea at present, the development level is still insignificant compared with advanced foreign countries, and the application range is very narrow depending on the antibiotic mechanism of microorganisms. Therefore, it is necessary to study the improvement of plant immune response by microorganisms as a solution to overcome this problem.

When the plant is infected with the disease, the host plant is determined by dividing the host plant into a friendly and unfriendly reaction in the interaction between the host plant and the plant pathogen at the early stage of the pathogen infection, by activating the defense mechanism in various intracellular metabolic pathways, (Defense) or not is determined.

In the unfriendly resistance response, infected plants recognize the invasion of pathogens, and in response to hypersensitive response such as local cell death, accumulation of callose, lignin, , And the like, and produces a variety of antimicrobial substances such as phytoalexin and produces a defense-related protein called pathogenesis related (PR) protein . These plant defenses are not only caused by pathogens, but also by artificial stimulation such as ethylene or methyl jasmonate, and are also reported to be caused by environmental stresses such as physical scars, salinity, and temperature.

If the plant's immune response is activated by microorganisms, plants responding to various categories of threats will initially be able to build defense systems and suffer less from disease and stress.

There have been disclosed microorganisms that have an effect of controlling plant diseases by inducing plant immunity reaction in the past (a method of inducing plant immunity through seed priming of a high temperature treated Bacillus culture solution, Korean Patent No. 1486708) There is no disclosure of microbial preparations for controlling plant diseases using randensis strains.

Thus, the present inventors have found that Erwinia < RTI ID = 0.0 > gerundensis KUDC9201 activates the defensive gene of the plant to control the plant disease, so that the strain can be used as a microorganism preparation for controlling plant diseases.

The present inventors have made efforts to develop a microorganism preparation for controlling environment-friendly plant diseases that can solve the problems of conventional organic or chemical fertilizer. As a result, it has been found that the Erwinia gerundensis KUDC9201 strain of the present invention has a plant disease control effect The present invention has been completed.

Accordingly, it is an object of the present invention to provide an Erwinia gerundensis strain KUDC9201 having a plant disease control effect and a microbial 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.

According to an aspect of the present invention as described above, the present invention is to freeze Winiah having a plant disease controlling effect reonden sheath (Erwinia gerundensis KUDC9201 strain (KCTC 13444BP).

In one embodiment of the present invention, the plant disease may be a scab disease of tobacco or a bacterial spot disease of pepper.

In another embodiment of the present invention, the strain may increase the expression level of a defensive gene of pepper to control bacterial foliar disease.

In another embodiment of the present invention, the defensive gene of the pepper is selected from the group consisting of Capsicum annuum Pathogenesis-related protein 4 (CaPR4) or Capsicum annuum Chitinase class 2 (CaChi2) Lt; / RTI >

In addition, the present invention relates to the aforementioned Erwinia < RTI ID = 0.0 > there is provided a microorganism preparation for controlling plant diseases, which contains at least one selected from the group consisting of strain KUDC9201, a culture of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof .

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.

According to the present invention, Erwinia < RTI ID = 0.0 > gerundensis strain KUDC9201 has the effect of controlling the bacillary spot disease of cigarette smoke or red pepper and that the strain increases the expression level of CaPR4 or CaChi2 which is a defense gene of pepper to control bacterial spotty disease of pepper, The Erwinia gerundensis KUDC9201 strain of the present invention is expected to be useful as an environmentally friendly biocontrol agent for promoting plant immunity by activating plant defense genes.

1 is a view to freezing Winiah according to one embodiment of the present invention showing a cigarette bottle sores bangjeryeok test result by the reonden sheath (Erwinia gerundensis) KUDC9201 strain.
FIG. 2 is a graph showing the results of a test for controlling bacterial blisters against red pepper caused by Erwinia gerundensis KUDC9201 strain according to an embodiment of the present invention.
Figure 3 is a view of the protective gene expression level changes in pepper by Earl Winiah to reonden sheath (Erwinia gerundensis) KUDC9201 strain in accordance with an embodiment of the invention.
4 is a view showing a 16S rDNA base sequence of Erwinia gerundensis KUDC9201 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.

It is an object of the present invention to provide an Erwinia gerundensis KUDC9201 strain (KCTC 13444BP) having a plant disease control effect.

In the present invention, the 16S rDNA nucleotide sequence of the strain was obtained and sequenced through Ezbiocloud (http://www.exbiocloud.net). As a result, the strain was identified as Earwinia germanstan, Erwinia gerundensis KUDC9201 strain was deposited with KCTC of Korea Biotechnology Research Institute on Jan. 2, 2018 and granted accession number KCTC 13444BP.

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

The plant disease may be a scab disease of tobacco or a bacterial spot disease of pepper.

The above Erwinia It was confirmed that the tobacco treated with gerundensis KUDC9201 showed high resistance to scab disease (see FIG. 1 and Experiment 1), and the pepper treated with the strain showed high resistance to bacterial spotty disease 2 and Experimental Example 2).

Sores disease of the cigarette in the present invention is a pathogen of Peg tobak Te Solarium Caro Caro Sat Sat boreom boreom (Pectobacterium carotovorum subsp. Carotovorum) (or El Winiah Caro Sat Sat Bora Bora Caro (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.

In the present invention, the bacterial fungus disease of the pepper is caused by the fungus, such as Zanthomonas campestris pv. Plague is caused by Kam lease (Xanthomonas campestris pv. Campestris), the pathogen penetrates the radish, cabbage, cauliflower, turnips, bok choy and many leaves, stems, and flower stalk and roots of cruciferous crops, causing a black bubyeong.

The bacterial spotted fever disease of the red pepper is a sign of a disease, and stains appear on leaf, petiole, stem, stem and fruit of pepper. The bacterial fungus of the pepper exhibits a dark brown or amorphous small spot on the leaf of the pepper at first and a yellowish halo around the dark brown spot.

When the bacterial fungus of the pepper develops, the central part of the lesion changes to white, and the edge of the lesion becomes brown or dark brown, and several lesions are combined to form a large lesion. When it gets worse, the leaves of the pepper die and the leaves of the pepper are formed. Also, a small spot of dark brown water-like shape is formed on the stem of the red pepper, and the central part of the lesion becomes convex later.

On the other hand, the above-mentioned Erwinia As a result of the reverse transcriptase polymerase chain reaction (RT-PCR) analysis of the degree of activation of the defensive gene of pepper treated with KUDC9201 strain, the strain was found to have a capsidum annuum Pathogenesis -related protein 4, CaPR4), and capsicum annuum Chitinase class 2 (CaChi2) gene of pepper (Fig. 3 and Experiment 3).

Thus, the present invention is to freeze Winiah reonden sheath (Erwinia gerundensis) KUDC9201 strain, the culture, the culture of a concentrate with water, the culture of water and Structures, and microbial agents and fertilizers, plant diseases, which comprises at least one selected from the group consisting of the active ingredient of the above strain and 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, red pepper and the like, 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 TSA (Tryptic soy agar) culture medium or LA (Luria-bertani agar) culture medium, 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 - Defender small  Fungi inducing Earl Winia Gerundensis  Isolation and Identification of KUDC9201

The strain KUDC9201 of Earwinia gerendensis according to the present invention was deposited at the genetic circle center of the Korea Institute of Science and Technology, Biotechnology Research Institute, which is a depository of the Korean patent strain on January 02, 2018 (Accession No .: KCTC 13444BP).

In the process of isolating the strain, the roots collected from the Dokdo Island were washed with sterile distilled water, and 100 μl of the washed root was transferred to a 1/10 TSA solid medium, which was a nutrient medium, using a micropipette. And cultured at 30 ° C for 24 hours. This process was repeated several times until the isolates of the isolates were isolated, and the isolates were purified. For the identification of the isolate, the base sequence of 16s rDNA of the strain was obtained and sequenced through Ezbiocloud (http://www.exbiocloud.net). As a result, the strain was identified as Erwinia gerendensis and named Erwinia gerundensis strain KUDC9201.

The 16S rDNA nucleotide sequence of the strain of the present invention, Earwinia gerendensis KUDC9201, is shown in FIG. When cultured in TSA (Tryptic Soy Agar) and LA (Luria-Bertani Agar) medium at 30 ° C for 12 hours, the strain forms a yellowish colony and sufficiently covers the TSA outlet surface after 24 hours of incubation.

Experimental Example 1 . Earl Winia Gerundensis KUDC9201  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. The strain KudC9201 isolated from Example 1 was cultivated in TSA medium for 24 hours, and was then dissolved in sterilized saline to adjust the concentration to 10 ⁸ CFU / ml. 20 쨉 l of the strain was dropped in each rhizome of the plant, And cultured at 25 ° C for 5 days. 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, Earwinia entericus strain KUDC9201 exhibited a high induction-resistance mechanism against tobacco rash disease, compared with the sterilized saline solution used as a negative control.

Experimental Example 2 . Earl Winia Gerundensis KUDC9201  Effect of Strain Treatment on Control of Bacterial Spotty Disease in Pepper

The pathogens used in this experiment were as follows: Mantus monas camphistris pv. Xanthomonas campestris pv. campestris) it was used KACC11158 strain, the pathogen was used as received from the pre-sale agricultural genetic resources center. Preparation of pathogens is done by Mantas Monas Campestris pv. Camphestris KACC11158 strain was plated on Yeast-Glucose Agar (YGC), cultured for 24 hours, and diluted with distilled water to a concentration of 10 ⁶ CFU / ml.

In this experiment, pepper seeds were sterilized in 1.2% sodium hypochlorite for 30 minutes, rinsed with sterilized water for 15 minutes, and seeds were transferred to a 50-well plug-containing plate containing 5 to 6 g of soil. The plants were cultivated in a plant culture room under the condition. After about 3 weeks, when the leaves reached about 4 leaves, the strain Earwienerendensis KUDC9201 isolated in Example 1 was dissolved in sterilized distilled water, and the concentration was adjusted to 10 ⁸ CFU / ml, . After 7 days, the prepared pathogen suspension was inoculated with a needle on the back of a red pepper leaf using a syringe. Five days after the inoculation, the symptoms appearing on the leaves of the plants were divided into grades 0 to 5. Sterile distilled water was used as negative control in this experiment.

As a result, as shown in Fig. 2, the strain Earwinia gorrantensis KUDC9201 exhibited a high induction resistance-defense mechanism against bacterial spot foliar disease of pepper, compared to the case of using a negative control sterilized saline solution.

Experimental Example 3 . Earl Winia Gerundensis KUDC9201  Of the red pepper Defense mechanism  Activation

Preparation of pepper plants, inoculation preparation of Erwinia entericis KUDC9201, and pathogens were treated in the same manner as in Experimental Example 2 above. The degree of activation of the defensive gene of pepper was analyzed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for 1-3 days after inoculation. As a result, as shown in Fig. 3, it was confirmed that the strain Earwinia germanusis KUDC9201 of the present invention increased the expression level of the CaPR4 and CaChi2 genes, which are pepper defense genes, to control the bacterial spot flaccid disease of red pepper.

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

Access number: KCTC13444BP

Checked on: 20180102

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Erwinia gerundensis strain KUDC9201 strain having antifungal activity          against pathogens, and uses thereof <130> MP17-307 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1460 <212> DNA <213> Erwinia gerundensis KUDC9201 16S rDNA <400> 1 cagattgaac gctggcggca ggcctaacac atgcaagtcg aacggtagca cagaggagct 60 tgctccttgg gtgacgagtg gcggacgggt gagtaatgtc tggggatctg cccgatggag 120 ggggataacc actggaaacg gtggctaata ccgcataacg tcgcaagacc aaagtggggg 180 accttcgggc ctcacaccat cggatgaacc cagatgggat tagctagtag gtggggtaac 240 ggctcaccta ggcgacgatc cctagctggt ctgagaggat gaccagccac actggaactg 300 agacacggtc cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag 360 cctgatgcag ccatgccgcg tgtatgaaga aggccttcgg gttgtaaagt actttcagcg 420 gggaggaagg ggacgaggtt aataacctcg ttcattgacg ttacccgcag aagaagcacc 480 ggctaactcc gtgccagcag ccgcggtaat acggagggtg caagcgttaa tcggaattac 540 tgggcgtaaa gcgcacgcag gcggtctgtt aagtcagatg tgaaatcccc gggcttaacc 600 tgggaactgc atttgaaact ggcaggcttg agtcttgtag aggggggtag aattccaggt 660 gtagcggtga aatgcgtaga gatctggagg aataccgggt ggcgaaggcg gccccctgga 720 caaagactga cgctcaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag 780 tccacgccgt aaacgatgtc gacttggagg ctgtgagcat gactcgtggc ttccggagct 840 aacgcgttaa gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga 900 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta 960 cctgctcttg acatccacgg aattcggcag agatgcctta gtgccttcgg gaaccgtgag 1020 acaggtgctg catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac 1080 gagcgcaacc cttatccttt gttgccagcg attcggtcgg gaactcaaag gagactgccg 1140 gtgataaacc ggaggaaggt ggggatgacg tcaagtcatc atggccctta cgagcagggc 1200 tacacacgtg ctacaatggc gcatacaaag agaagcgacc tcgcgagagc aagcggacct 1260 cacaaagtgc gtcgtagtcc ggatcggagt ctgcaactcg actccgtgaa gtcggaatcg 1320 ctagtaatcg tggatcagaa tgccacggtg aatacgttcc cgggccttgt acacaccgcc 1380 cgtcacacca tggggagtgg gttgcaaaag aagtaggtag cttaaccttc gggagggcgc 1440 ttaccacttt gtgattcatg 1460

Claims (7)

Erwinia with plant disease control effect ( Erwinia gerundensis strain KUDC9201 (KCTC 13444BP).
The method according to claim 1,
Characterized in that the plant disease is scurf disease of tobacco or bacterial spotty disease of pepper, Erwinia &lt; RTI ID = 0.0 &gt; gerundensis ) KUDC9201 strain.
The method according to claim 1,
The strain increases the expression of defense genes in pepper, characterized in that to control the bacterial jeommunuibyeong, Earl Winiah to reonden sheath (Erwinia gerundensis ) KUDC9201 strain.
The method of claim 3,
Characterized in that the defensive gene of the pepper is Capsicum annuum Pathogenesis-related protein 4 (CaPR4) of pepper or Capsicum annuum Chitinase class 2 (CaChi2) of pepper. Erwinia gerundensis strain KUDC9201.
A method for producing a recombinant yeast strain selected from the group consisting of Erwinia gerundensis KUDC9201 strain according to any one of claims 1 to 4, a culture of the strain, a concentrate of the culture, a dried product of the culture, A microorganism preparation for controlling plant diseases, comprising at least one active ingredient.
A microbial fertilizer for controlling plant diseases, comprising the microbial agent of claim 5.
A method for controlling plant diseases, comprising treating the microbial agent of claim 5 with a soil, a plant, or a seed of a plant.

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