KR101711091B1 - Endophytic Variovorax paradoxus KB5 strain having controlling activity against plant pathogen and uses thereof - Google Patents

Abstract

The present invention relates to a method for inhibiting plant pathogens, which comprises an endogenous bacterium of the genus Variovorax paradoxus KB5 having a controlling activity against a plant pathogenic bacterium that causes plant diseases, a plant pathogen control composition containing the strain or a culture thereof as an active ingredient, A method for controlling a plant pathogenic bacterium comprising the step of cultivating a strain and a method for treating a plant pathogenic bacterium comprising the step of treating an effective amount of the strain or a culture thereof with a crop, a seed or a cultivation field of a crop, The present invention relates to a strain of KB5 strain, which has an advantage of being able to have a beneficial effect on plants constantly in the process of interacting with plants through symbiosis with plants. Especially, when a plant disease occurs, Inhibitory effect can be more effectively exhibited, and a microorganism for biological control Relates to verifying that the zero strain useful information.

Description

[0001] The present invention relates to a bacterium belonging to the genus Boryovarax paradoxus, which is an endogenous bacterium having a controlling activity against a plant pathogenic bacterium,

The present invention relates to an endogenous bacterium, Pariboxaparas parahaemolyticus KB5, which has an antifungal activity against a plant pathogenic bacterium, and its use, and more particularly to a bacterium belonging to the genus Bariobacter A method for controlling plant pathogens, comprising the step of cultivating the strain, and a method for producing the effective amount of the strain or the culture solution thereof, A method for controlling a plant pathogenic bacterium comprising the step of treating a crop, a seed or a cultivation land of a crop.

Creatures are symbiotic with many other creatures in the ecosystem. Plants are also symbiotic with a variety of fungi in soil and in the atmosphere, one of which is endophytes present in plant tissues. Mycotrophs live in plants without any negative symptoms in plants, and have beneficial effects on plants in their interaction with plants. Until now, it has been found that a variety of endogenous fungi grow in many kinds of plants. Among them, it provides a special substance to the plant through its own metabolism or induces a specific metabolism of the plant, . It also exhibits antimicrobial activity through the various substances produced in the process of interacting with plants and their efficacy. Mycotoxins found in some herbaceous plants play a role in protecting plants from weeds and pathogens, and some live organisms increase the resistance of plants to illness and environmental stress. In addition, mycotoxins produce antimicrobial and antioxidant substances. In recent research, 51% of the newly reported biologically active substances have been isolated from endogenous organisms, and they are expected to solve the difficulties of finding new substances. Thus, mycotoxins have diverse potential usefulness as genetic vectors, secondary metabolite producers, and biological control agents, and their importance is increasing.

Investigating the diversity of these endemic bacteria and studying their roles in the inside and outside of the plant suggests the possibility of biological control of various diseases and it is a good method to be used for development and use as a microbial preparation. In addition, many useful substances such as biologically active substances, growth regulators for animals and plants, and precision biochemicals are searched from microorganisms and utilized in industrial fields such as pharmaceuticals, food and agriculture, environment, resources and energy, and pharmaceuticals and various high value- And the width of development is getting wider.

Korean Patent Publication No. 2010-0061338 discloses a method for controlling flora and plant disease, and Korean Patent No. 1270664 discloses a method for controlling phytopathogenic bacteria and fungi and a method for inhibiting multidrug- Discloses Bacillus amyloliquefaciens EML-JUN11, which is an endophytic bacterium having an antifungal activity against plant pathogenic bacteria, as disclosed in the present invention. Not at all.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs. In the present invention, Bari Ovarax paradoxus KB5 strain was isolated and identified from endemic bacteria existing on the leaves at the onset of the disease of Arabidopsis thaliana. ( Pseudomonas syringae ). ≪ / RTI >

Therefore, the B. paroxyspora KB5 strain isolated through the present invention is an endogenous strain and can more effectively exhibit the antagonistic effect of inhibiting the growth of plant pathogenic bacteria occurring in plants, and thus can be very usefully used as a biological microorganism preparation The present invention has been completed.

In order to achieve the above object, the present invention provides a strain of Variovorax paradoxus KB5, which is an endophyte (Endophytes) having a controlling activity against a plant pathogenic bacterium producing a plant disease.

In addition, the present invention provides a composition for inhibiting plant pathogens, comprising the above-mentioned strain Variovorax paradoxus KB5 or a culture thereof as an active ingredient.

In addition, the present invention provides a method for preparing a composition for controlling plant pathogenic bacteria, which comprises culturing the strain Variovorax paradoxus KB5.

In addition, the present invention provides a method for controlling a plant pathogenic bacterium, which comprises treating the effective amount of the above-mentioned Variovorax paradoxus KB5 or a culture thereof with a crop, a seed of a crop or a cultivation field.

The baryovirus paradoxus strain KB5 of the present invention is an endogenous strain having an advantage that it can have a useful effect on plants constantly in the process of interacting with plants through symbiosis with plants. In particular, Can effectively exhibit the antagonistic effect of inhibiting growth, and can be very useful as a microbial preparation for biological control. In addition, the microorganism preparation containing the strain of the present invention as an active ingredient can be used as an environmentally friendly biological pesticide free from the risk of environmental pollution and human toxicity due to residual pesticides caused by the side effects of pesticides, It will be possible.

Figure 1 shows the Arabidopsis ( Arabidopsis < RTI ID = 0.0 > thaliana ) plants from the uninoculated leaves.
Fig. 2 shows the result of re-isolation of the isolate endovagent, Variovorax paradoxus KB5 strain, on the leaves of the Arabidopsis root again. (A) Treating distilled water (DW) and Arabidopsis parahaemolyticus KB5 (KB5) which is an endogenous bacterium isolated in the present invention, respectively, in a root of Arabidopsis thaliana, (B) It is the result of confirming whether the lactus parasiticus KB5 strain is re-separated or cultured.
Fig. 3 is a graph showing the results obtained when the baryovirus paradoxus KB5 of the present invention is Pseudomonas sp. syringae ) was analyzed by GFP expression level.
4 shows the disease resistance effect (disease incidence (%)) that occurs when treating the root of Arabidopsis thaliana planted with Pseudomonas syringae with the treatment of Bariophorus paradoxus KB5 of the present invention .

In order to accomplish the object of the present invention, the present invention provides Variovorax paradoxus KB5 strain, which is an endophyte having an antifungal activity against plant pathogenic bacteria causing plant diseases.

The strain of the present invention was isolated from the endogenous bacteria existing on the leaves of Arabidopsis thaliana infected with plant pathogens. A single colony was taken for the separated microorganisms and treated again on the root of Arabidopsis thaliana. Leaves were collected again and plated on LB agar plates And confirmed that the microorganisms were resuspended in the actual Arabidopsis. From the homology analysis of the 16s rRNA sequence of the isolate endogenous KB5, KB5 was found to be more than 99% identical to that of Variovorax paradoxus, and it was confirmed that KB5 Lt; / RTI > The strain exhibited an inhibitory activity against Pseudomonas syringae in plants. The strain was deposited on April 15, 2015 by the Korea Research Institute of Bioscience and Biotechnology (KCTC) (Deposit No. KCTC18377P).

In a strain according to an embodiment of the present invention, the plant pathogen is a Pseudomonas sp. sp.) can hospital uniform and, preferably Pseudomonas ache dog (Pseudomonas syringae , but are not limited thereto.

In addition, the present invention provides a composition for inhibiting plant pathogens, comprising the above-mentioned strain Variovorax paradoxus KB5 or a culture thereof as an active ingredient. The composition can prevent, but is not limited to, stem sickness, bacterial blight, and the like caused by Pseudomonas sylin.

The composition for controlling a plant pathogenic bacterium of the present invention may be a formulation in the form of a wettable powder or a suspension concentrate. The controlling composition according to the present invention may be prepared in the form of a liquid sterilizing agent, and may be added in the form of powders by adding an extender, or it may be granulated by formulating it into a powder. However, the formulation is not particularly limited. In other words, it can be formulated as a bio-disinfectant to overcome this in environment-friendly organic farming with limited supply of chemical disinfectants.

The wettable powder of the present invention may be prepared by mixing, as an active ingredient, Variovorax paradoxus KB5 strain or culture thereof, white carbon as a moisture absorbent, sodium bis [2-ethylhexyl] sulfosuccinate as a wetting agent Sodium lignosulfonate as a dispersing agent and kaolin as an extender, preferably 10% by weight of a strain of Variovorax paradoxus KB5 or a culture thereof, 1% by weight of white carbon, But are not limited to, 1 wt% sodium bis [2-ethylhexyl] sulfosuccinate, 1 wt% sodium lignosulfonate, and 87 wt% kaolin.

The liquid wettable powder of the present invention can be used as an active ingredient of Variovorax paradoxus KB5 strain or as a culture fluid, wetting agent and dispersant for MBSC (Nonylphenol, ethoxylated, monoether with sulfuric acid, sodium salt, sodium bis [ 20 ethylhexyl] sulfosuccinate Polyoxyethylene nonylphenol), isopropanol as an excipient and water as an extender. The liquid wettable agent is preferably, but not limited to, 50% by weight of a strain of Variovorax paradoxus KB5 or a culture thereof, 4% by weight of MBSC, 30% by weight of isopropanol and 16% by weight of water.

In addition, the present invention provides a method for preparing a composition for controlling plant pathogenic bacteria, which comprises culturing the strain Variovorax paradoxus KB5. The method for culturing Variovorax paradoxus KB5 strain and the method for preparing a composition for controlling botanical pathogens can be any method known in the art and is not particularly limited to a specific method.

In addition, the present invention provides a method for controlling a plant pathogenic bacterium, which comprises treating the effective amount of the above-mentioned Variovorax paradoxus KB5 or a culture thereof with a crop, a seed of a crop or a cultivation field.

As a method for controlling the plant pathogenic bacterium, an effective amount of a culture medium obtained by cultivating the strain Variovorax paradoxus KB5 may be treated to a crop, a seed of a crop or a cultivation land.

The " effective amount " of the present invention means that the control composition is uniformly diluted with water to control plant pathogens in an amount sufficient to produce beneficial or desired results, and then sprayed onto the plant and arable land using a suitable spraying device such as a power sprayer . When the wettable powder or the liquid wettable powder of the present invention is diluted in water, the concentration of the wettable powder or the liquid wettable powder is adjusted to about 10 ⁵ to 10 ¹⁰ cfu / ml, preferably about 10 ⁸ cfu / ml so that the effective ingredient can be in a biologically effective range But is not limited thereto.

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. Plants Of my live bacteria  Isolation and sequencing

In order to select the live bacteria in the plant which can be used as a microorganism preparation by enhancing the disease resistance to the plant, the endogenous bacteria existing on the leaves of Arabidopsis thaliana, which is known as a model plant, were isolated. Pseudomonas syringae , a plant pathogenic bacterium, was inoculated on the leaves of Arabidopsis thaliana and microorganisms presumed to be endogenous bacteria were isolated from apoplast fluid of uninoculated leaves (Fig. 1). A single colony was taken for the separated microorganisms and treated again on the root of Arabidopsis. After collecting the leaves again, it was confirmed that the treated microorganisms were re-separated when they were applied to the LB agar plate, (Fig. 2). A homology analysis of the 16s rRNA sequence of the isolated endogenous KB5 showed that KB5 was more than 99% identical to that of Variovorax paradoxus (Table 1).

Systematic analysis of mycobacteria KB5 Phylum Strain Bacterial species 16S rRNA  ( bp ) Proteobacteria KB5 Variovorax paradoxus 990

Example  2. Of my live bacteria  Antimicrobial Activity Test

The live bacteria of the plant, Biolobolax paradoxus KB5Variovorax paradoxus KB5) was used as a phytopathogenic bacterium, Pseudomonas sylinPseudomonas syringae) Were investigated for their antibacterial activity. Pseudomonas sylin (Pseudomonas syringae) And Bari ovorax paradoxasus KB5Variovorax paradoxus KB5) at the same concentration were cultured in LB agar plates, and the suspension of Pseudomonas sylinPseudomonas syringae) Was used to confirm the degree of growth. As a result, Barrio vulgaris paradoxasus KB5Variovorax paradoxus KB5) is a pseudomonas syllin (Pseudomonas syringae) (Fig. 3).

Example  3. Of my live bacteria  Cultivation prize Protection  Confirm

My live bacteria Bariobolax paraffinosus KB5 ( Variovorax paradoxus KB5) were tested in real plants in order to evaluate the antibacterial activity of the plants in Arabidopsis thaliana. The root of the Arabidopsis thaliana was treated with Variovorax paradoxus KB5, followed by Pseudomonas sp. syringae ) was treated and the disease was induced. After 5 days, Bariovorax paradoxus KB5 ( Variovorax paradoxus KB5). As a result, Variovorax KB5 paradoxus KB5) showed about 70% incidence rate in the control group, whereas the endogenous control group had a disease incidence rate of 40% or less in the control group. Pseudomonas spp. syringae ) (Fig. 4).

From the above results, it was confirmed that Variovorax paradoxus KB5, a live bacterium in the plant, exhibited excellent disease inhibition rate against plant pathogens. Through the test of potency in actual Arabidopsis, KB5 ( Variovorax paradoxus KB5) to develop microbial agents.

Korea Biotechnology Research Institute KCTC18377P 20150415

<110> Korea Research Institute of Bioscience and Biotechnology <120> Endophytic Variovorax paradoxus KB5 strain having controlling          activity against plant pathogens and uses thereof <130> PN15133 <160> 1 <170> KoPatentin <210> 1 <211> 1414 <212> DNA <213> Variovorax paradoxus <400> 1 gcttacacat gcaagtcgaa cggcagcgcg ggagcaatcc tggcggcgag tggcgaacgg 60 gtgagtaata catcggaacg tgcccaatcg tgggggataa cgcagcgaaa gctgtgctaa 120 taccgcatac gatctacgga tgaaagcagg ggatcgcaag accttgcgcg aatggagcgg 180 ccgatggcag attaggtagt tggtgaggta aaggctcacc aagccttcga tctgtagctg 240 gtctgagagg acgaccagcc acactgggac tgagacacgg cccagactcc tacgggaggc 300 agcagtgggg aattttggac aatgggcgaa agcctgatcc agccatgccg cgtgcaggat 360 gaaggccttc gggttgtaaa ctgcttttgt acggaacgaa acggtctttt ctaataaaga 420 aggctaatga cggtaccgta agaataagca ccggctaact acgtgccagc agccgcggta 480 atacgtaggg tgcaagcgtt aatcggaatt actgggcgta aagcgtgcgc aggcggtgat 540 gtaagacagt tgtgaaatcc ccgggctcaa cctgggaact gcatctgtga ctgcatcgct 600 ggagtacggc agagggggat ggaattccgc gtgtagcagt gaaatgcgta gatatgcgga 660 ggaacaccga tggcgaaggc aatcccctgg gcctgtactg acgctcatgc acgaaagcgt 720 ggggagcaaa caggattaga taccctggta gtccacgccc taaacgatgt caactggttg 780 ttgggtcttc actgactcag taacgaagct aacgcgtgaa gttgaccgcc tggggagtac 840 ggccgcaagg ttgaaactca aaggaattga cggggacccg cacaagcggt ggatgatgtg 900 gtttaattcg atgcaacgcg aaaaccttac ccacctttga catgtacgga attcgccaga 960 gatggcttag tgctcgaaag agaaccgtaa cacaggtgct gcatggctgt cgtcagctcg 1020 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgtcatt agttgctaca 1080 ttcagttggg cactctaatg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140 caagtcctca tggcccttat aggtggggct acacacgtca tacaatggct ggtacaaagg 1200 gttgccaacc cgcgaggggg agctaatccc ataaaaccag tcgtagtccg gatcgcagtc 1260 tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgt ggatcagaat gtcacggtga 1320 atacgttccc gggtcttgta cacaccgccc gtcacaccat gggagcgggt tctgccagaa 1380 gtagttagct taaccgcaag gagggcgata ccac 1414

Claims (7)

Variovorax paradoxus KB5 strain (KCTC18377P), an endophyte having an antifungal activity against Pseudomonas syringae pathogen. delete delete delete A composition for controlling Pseudomonas syringae pathogen comprising the Variovorax paradoxus KB5 strain (KCTC18377P) or a culture thereof according to claim 1 as an active ingredient. A method for preparing a composition for controlling Pseudomonas syringae pathogen, comprising culturing the Variovorax paradoxus KB5 strain (KCTC18377P) according to claim 1. A method for controlling Pseudomonas syringae pathogens, which comprises treating an effective amount of a strain of Variovorax paradoxus KB5 strain (KCTC18377P) or a culture thereof according to claim 1 to a crop, a seed or a cultivation field of a crop, How to.

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