KR20180117322A - A novel Serratia sp. IM2, and Composition for plant disease control comprising the same - Google Patents

Abstract

The present invention relates to a serratia sp. IM2 strain (deposit number: KACC 92171P) having a disease control activity against at least one plant pathogen selected from plant pathogens comprising Xanthomonas campestri and Erwinia carotovora, and a composition comprising the strain. A strain of the present invention has a biological disease control ability against the bacterial spot (Xanthomonas campestris pv. vesicatoria) and the bacterial soft rot (Erwinia carotovora carotovora SCCI), thereby being used in controlling the diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel Celastia espe IM2 strain, IM2, and Composition for plant disease control comprising the same}

The present invention relates to a novel Celastia espe IM2 strain and a composition for controlling a plant disease containing the same.

Organic pesticides, which are mainly used as agrochemicals, are effective in small amounts and have a great role in increasing the income of farm households because they are inexpensive. However, many of the organic pesticides that have been used in the past have been limited in their use due to the possibility of cancer induction or the residual toxicity, and the serious side effects such as increasing the resistance of the plant pathogens to organic pesticides, acidification of soil, destruction of ecosystem . In recent years, as a countermeasure against these environmental pollution and residual toxicity, new pesticides and fertilizers have been researched and developed, and studies on natural microbial agents having a good decomposition and weak toxicity have been actively carried out.

Biological control using microorganisms uses antibiotics or toxins produced from soil microorganisms to protect plants from infection by microbial pesticides that control pathogens or insect pests or by antagonism of microorganisms themselves and indirectly promote plant growth And microbial fertilizers. Biological control methods using antagonistic microorganisms include chitin, chitinase capable of degrading cell wall components such as chitin, β-1,3-glucan, and mannan, Glucanase, mannase, and antibiotic mechanism which directly inhibits the growth of phytopathogenic fungi by secreting antibiotic substance which is a specific metabolite of antagonistic microorganism.

Biological pesticides include microbial pesticides that use microorganisms to control plant diseases, pests and weeds, and biochemical pesticides that use natural products produced by plants or microorganisms. In the case of microbial pesticides, it is difficult to mass-produce and formulate products, and since the storage stability of products is low, standardization, standardization, and ease of distribution are not secured like chemical pesticides, and the market has not been greatly expanded. In addition, when the microbial pesticide is used in actual packaging, the plant disease can be controlled only after the microbes have grown to a considerable amount after the treatment, so that it is difficult to control the plant disease rapidly. In addition, since the spectrum of applied diseases is narrow, various bio-pesticides must be treated in order to control other plant diseases occurring in the crops, which realistically puts an economic burden on the farmers.

On the other hand, bacteria of the genus Bacillus, which are mainly present in the soil rhizosphere, produce various secondary metabolites showing antimicrobial activity such as sufectin and iturin, and are resistant to Gram-positive It is a germ. In addition, since most strains of Bacillus subtilis are safe against phosphorus and toxicity, many studies have been carried out to develop biological control agents using them.

In Korea, Bacillus amyloliquefaciens LP03 strain (Korean Patent No. 0807403), Bacillus atrophares CNU05-1 strain (Korean Patent No. 0773091), and Bacillus amyloliquefaciens strain (Bacillus amyloliquefaciens strain) as a strain for controlling the gray mold of Bacillus sp. Bacillus subtilis KCCM10639 or KCCM10640 strain (Korean Patent No. 0767437) for plant disease control has been registered.

However, a number of microbial control agents exhibiting antifungal activity as the above-mentioned Bacillus sp. Strain have been disclosed, but they have not satisfied the need for bio-pesticides capable of effectively controlling various plant diseases. Therefore, Is required.

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and it is an object of the present invention to provide a strain having high antibacterial activity against red pepper bacterial spotty disease and cabbage blight.

It is another object of the present invention to provide a method for controlling against bacterial spotty fever of pepper and Chinese cabbage blight.

In order to accomplish the above object, the present invention relates to a method for producing Xanthomonas campestri pv . Serratia sp.) IM2 strain (Accession No. KACC 92171P), which has a controlling activity against at least one plant pathogenic bacterium selected from plant pathogens consisting of vesicatoria and Erwinia carotovora carotovora .

In addition, the present invention provides a composition for controlling at least one plant disease selected from the group consisting of bacterial spotted fever disease and rotifer comprising the strain Serratia sp. IM2 (Accession No. KACC 92171P) or a culture thereof as an active ingredient.

In one embodiment of the present invention, the plant is preferably pepper or cabbage, but is not limited thereto.

The present invention also relates to a method for the treatment of bacterial spotted febrile diseases and blisters in a plant characterized by the treatment of a composition comprising the Serratia sp. IM2 strain (Accession No. KACC 92171P) or a culture thereof in an environment in which the plant or the plant grows At least one plant selected from the group consisting of plants.

In another embodiment of the present invention, the method of treatment is a method of treating Serratia sp. IM2 strain (Accession No. KACC 92171P) in an amount of 1 × 10 ⁵ cfu / ml to 1 × 10 ⁸ cfu / ml But is not limited thereto.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for the preparation of Xanthomonas < RTI ID = 0.0 > campestri pv . vesicatoria and Erwinia (Accession No. KACC 92171P) having a controlling activity against at least one plant pathogenic bacterium selected from plant pathogens composed of a plant belonging to the genus Pseudomonas aeruginosa , Carotovora carotovora .

More specifically, the Serratia sp. IM2 strain according to the present invention was obtained by isolating a sample from a larva of a dark blue leaf worm and then analyzing the base sequence of the 16S rDNA of the strain. As a result, Serratia sp. sp.). The new strain was designated as Serratia sp. IM2 strain and deposited at the National Institute of Agricultural Science and Technology on March 27, 2017, and received the deposit number KACC 92127P. Please refer to Fig. 1 and Fig.

A composition comprising the Serratia sp. IM2 strain according to the present invention (accession number KACC 92171P) itself, its culture or an extract of the culture may be used in an environment in which the plant is growing (e.g. soil, water) The plant can be biologically controlled by spraying or treating the surface of the plant in an amount that can inhibit the plant disease. In this case, if necessary, it may be mixed with a carrier commonly used in the field of agrochemicals, and powder, pellets, Or a solution or the like to be used as a composition for controlling plant diseases. The carrier may be water, white carbon, kaolin or the like, and the culture may be a liquid culture or a solid culture.

When the composition for controlling plant diseases according to the present invention is packaged, the concentration of the strain in the composition may vary depending on the density of pathogens of the packaging, the onset condition, etc. However, the concentration of the strain may be in the range of 1 × 10 ⁵ cfu / ml to 1 × 10 ⁸ cfu / , And particularly exhibits excellent control effect at a concentration of 1 x 10 < ⁸ > cfu / ml.

The composition according to the present invention may be applied to soil, crop leaves, stems, flowers or fruits by diluting the composition in water. In order to add the antimicrobial activity of the microorganism strain of the present invention A foliar spraying agent, a cross-hair spraying agent, and the like. In addition, it is possible to further increase the control effect by pre-treating the sprayer before the onset and proliferation of a plant pathogenic bacterium.

The novel strain of Celastia espe IM2 according to the present invention is a strain of Xanthomonas campestri pv . vesicatoria) and cabbage blight ( Erwinia carotovora carotovora SCCI) have biological control ability and can be used to control these diseases.

1 shows the nucleotide sequence of the 16S rDNA of the SELATIA ASPY IM2 strain according to the present invention,
FIG. 2 is a result of confirming the phylogenetic position of 16S rDNA of the novel Celastia espe IM2 strain according to the present invention,
Figures 3 and 4 illustrate the novel Serratia sp. FIG. 3 shows the cellulase production, FIG. 4 shows the result of confirming the protease production ability,
Figure 5 shows the results of a novel Serratia < RTI ID = 0.0 > sp. The antimicrobial activity of Erwinia carotovora carotovora SCCI and Xanthomonas campestri pv . Vesicatoria were investigated in IM2 strain .
Figure 6 shows that Serratia < RTI ID = 0.0 > sp . Bacterial Spotty Disease of the IM2 Strain ( Xanthomonas campestri pv . vesicatoria) and Erwinia carotovora carotovora (SCCI).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it should be understood that the present invention is not limited by the following examples, and that various modifications or changes may be made therein without departing from the spirit and scope of the present invention.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Example  1: Selatia Espie  Isolation and Identification of IM2 Strain

After a sample is isolated from a larva of a navy blue leaf worm, 1 g of each sample is put into sterilized water and shaken at room temperature. The shaken solution is diluted 10-fold with sterilized water, and then the dilution suspension is plated in 100 쨉 l of various media and cultured at 25 째 C for 48 hours. The isolated strains were examined for their antibacterial and antimicrobial activities and selected strains having excellent ability.

16S rDNA sequencing was performed for the molecular genetic analysis of the selected strains. Based on the results, homology with NCBI GeneBank database was searched and CLASTAL X program and PHYLIP program were used to confirm the phylogenetic position.

Analysis of the 16S rDNA sequence (Figure 1) of the selected strains showed 99% homology with the Serratia nematodiphila strain. From the results of this analysis, it was confirmed that the strain belongs to Serratia genus 2)

The above-mentioned isolated strain was named Serratia sp. IM2, and deposited it with the Center for Agricultural Genetic Resources, National Institute of Agricultural Science and Technology.

The composition of the various media used in the present invention is as follows.

a. 1/3 KB (proteose peptone _{# 3, 6.7g, K 2 HPO} 4, 0.5g, MgSO 4 · 7H 2 O, 0.5g, glycerol, 5ml, agar, 15g / 1L of distilled water)

b. 1/10 TSA (Pancreatic digest of casein 1.5 g, Papaic digest of soybean 0.5 g, NaCl 0.5 g, agar 15 g / 1 L distilled water)

c. LM (peptone 5g, MgSO _{4 ·} 7H ₂ O 0.2g, Ferric ammonium citrate 0.1g, 0.05g CaCl _2, FeCl _{3 ·} 6H ₂ 0 0.01g, 0.01g MnSO _{4 ·} 4H2O, agar 15g / 1L of distilled water)

d. R2A (Proteose peptone 0.5 g, casamino acid 0.5 g, yeast extract 0.5 g, glucose 0.5 g, water-soluble starch 0.5 g, K ₂ HPO ₄ 0.3 g, MgSO ₄ .7H ₂ O 0.05 g, sodium pyruvate 0.3 g, 1 L distilled water)

e. NA (Nutrient broth 8 g, baby 15 g / 1 L distilled water)

f. _{RBA (K 2 HPO 4 1.0g,} MgSO 4 · 7H 2 O 0.5g, Bacto peptone 5g, glucose 10g, rose-bengal 0.003g, agar 15g / 1L of distilled water)

Example 2: Serratia sp . Enzyme activity of strain IM2

1) whether cellulase is produced

Serratia sp. IM2 strain is cultured in LB medium for 48 hours. The cells were then suspended in CMC agar medium (0.2% NaNO ₃ , 0.1% K ₂ HPO ₄ , 0.05% MgSO ₄ , 0.05% KCl, 0.2% carboxymethylcellulose sodium salt, 0.02% peptone, 1.7% agar / ㎕ is dropped and then cultured again. After incubation, immerse Gram's iodine solution (1 g KI, 0.5 g iodine / 150 ml distilled water) for 3 minutes to 5 minutes. Finally, cellulase production was confirmed according to whether Halo zone was generated or not (FIG. 3).

2) Protease production ability

Add 0.7% skim milk to the LB medium to make a protease-producing medium. 10 μl of the strain is dropped and cultured for 2 days. After culturing, the presence or absence of Halo zone formation was confirmed (FIG. 4).

Example  3: Anti-bacterial  Active test

Serratia sp. Chinese cabbage blight of strain IM2 ( Erwinia carotovora carotovora SCCI) and red pepper bacterial spotty disease ( Xanthomonas campestri pv . vesicatoria) were tested for anti-bacterial activity. Each pathogenic microorganism was plated on LB agar medium, and then Serratia sp. IM2 strain was incubated for 24 hours. As a result, it was confirmed that the growth of each bacterial pathogen was inhibited (FIG. 5).

Example 4: Plant bacterial Disease control  Effect test

Serratia sp . Bacterial Spotty Disease of Hot Pepper of IM2 Strain ( Xanthomonas campestri pv . vesicatoria) and Chinese cabbage rot ( Erwinia carotovora carotovora SCCI). Serratia sp . IM2 strain was cultured at 30 ℃ for 2 days and treated with sterilized water.

Bacterial spotty fever of pepper is Serratia sp . IM2 strain was sprayed with 4 x 10 ⁶ cfu / mL of pathogens. The inoculated pepper plants were transferred to a wet chamber and maintained at 25 ℃ and relative humidity of 100% in the dark condition. After 3 ~ 4 days, the incidence was confirmed. In case of cabbage smear, the tobacco was replaced with 12 - Serratia sp . IM2 strain. Two days later Serratia sp . IM2 strain was treated with 1 × 10 ⁶ cfu / mL of the Chinese cabbage isolate on the treated tobacco leaf, and the incidence was examined after 2 to 3 days (FIG. 6).

[Access number]

Depositor Name: Agricultural Biotechnology Research Institute

Accession number: KACC92171

Checked on: 20170327

Claims (5)

Xanthomonas campestri pv . vesicatoria and Erwinia carotovora carotovora (Accession No. KACC 92171P), which has a controlling activity against at least one plant pathogenic bacterium selected from plant pathogens. A composition for controlling at least one plant disease selected from the group consisting of Serratia sp. IM2 strain (Accession No. KACC 92171P) or bacterial spotty fever disease and blight disease containing the culture liquid as an active ingredient. The plant disease controlling composition according to claim 2, wherein the plant is pepper or cabbage. Characterized in that the composition comprising the Serratia sp. IM2 strain (Accession No. KACC 92171P) or a culture thereof is treated in an environment in which the plant or the plant is growing, wherein the plant is selected from the group consisting of bacterial floret disease and rotifer A method for controlling one or more plant diseases. 5. The method according to claim 4, wherein the plant is pepper or cabbage.

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