KR101403304B1 - Streptomyces acidiscabies ＪＡ（Ⅱ）-10, biopesticide comprising the strain against gray mold and control method of gray mold with same - Google Patents

Abstract

The present invention relates to the microbiological characteristics of Staphyllacesis systisvisis JA (II) -10 strains exhibiting an antibacterial activity against a strawberry gray mold pathogen. The microorganism characteristics of straptomyces strain JA (II) -10 can be used for the control of plant gray mold diseases by using microorganisms naturally grown in Korea, which can provide economical substitution effect, can be environmentally friendly and highly effective, and can increase the commerciality and productivity of crops The effect is great.

Description

TECHNICAL FIELD The present invention relates to a microorganism preparation for preventing strains of gray mold and a method for controlling gray mold disease, which contains the strain JA (Ⅱ) -10, a biopesticide containing the strain against gray mold and control method of gray mold with same}

The present invention relates to Staphylococcus epidermidis JA (II) -10 strain and a microorganism strain for controlling plant gray mold disease containing the same.

Gray mold fungus is a multipotent bacterium that occurs in almost all vegetables, flowers, and fruits in the ground. The bacterium also causes corruption in fruit and vegetables during storage, transportation and sale secondary to harvest.

Especially in recent years, resistance to chemotherapy has been decreasing due to the frequent appearance of resistant bacteria to existing chemical pesticides. In case of pesticides having some effects, The use thereof is strictly limited. Therefore, studies on the development of environmentally friendly biocidal pesticides that can replace chemical pesticides have been actively carried out.

Efforts to control gray mold as a biological pesticide have been proposed using natural products and microorganisms, and in particular, Bacillus sp. spp . ), Tri coder equine (Trichoderma spp.), Yeast (yeast) is a microbial pesticides have been developed to take advantage of the global registration and so on. In the case of foreign Pseudomonas syringe (Pseudomonas (Bio-save 10), which is a product of Bacillus syringae ( Bacillus sp. sp . ) Was commercialized as a gray mold disease and powdery mildew control agent in 2003. Even if your local bacillary piece FORT Miss to inhibit the mycelial growth and spore germination of Botrytis bacteria (Bacillus Research on gray mold disease control with licheniformis) N1 strain was reported.

However, despite many research examples, there is no established biocide for the control of gray mold disease, which is effective, inexpensive and safe. Therefore, it is required to search for a new microorganism genetic resource having excellent antimicrobial activity and stable characteristics as pesticide.

Accordingly, the present inventors have succeeded in separating microorganisms having antifungal activity by isolating strains of Staphylococcus aureus Esisseisvisis microorganism strains that are antagonistic to plant pathogens, and cultivating them in a large amount, into pathogenic fungi of major crops The present invention has been completed based on the discovery that strawberry gray mold disease can be efficiently controlled.

It is an object of the present invention to provide a novel microorganism having an excellent antifungal activity against a plant pathogenic bacterium of a crop and exhibiting an excellent controlling effect against a gray mold disease.

It is another object of the present invention to provide a microbial preparation for controlling a non-toxic environment friendly plant gray mold disease containing the novel microorganism and a method for controlling gray mold.

The present invention relates to the use of Streptomyces < RTI ID = 0.0 > acid < / RTI > bacteria microorganism strain and a microbial agent for controlling plant diseases using the same.

More specifically, the strains were isolated from the soymilk soil and designated as Staphyllaces siebisis JA (II) -10, and cultured in large quantities to treat the pathogenic fungi of the main crops to produce strawberry gray mold A microorganism preparation for controlling microorganisms and a method for controlling a gray mold disease using the microorganism preparation are provided.

Streptomyces < RTI ID = 0.0 > acidisabies JA (II) -10 (KACC 91492P) is a microorganism native to Korea and isolated from the soymilk soil. It is a microorganism isolated from Botrytis (Staphylococcus aureus) Cinerea ) is inhibited by mycelial growth.

In addition, Streptomyces < RTI ID = 0.0 > acidisabies JA (II) -10 (KACC 91492P) and a culture thereof are characterized by having a strong antagonistic action against a strawberry gray mold disease.

Hereinafter, the present invention will be described in detail.

The present invention provides Streptomyces acidiscabies JA (II) -10 (KACC 91492P) strain isolated from Syrup rhizosphere soil and having antifungal activity against plant gray mold pathogens.

More specifically, the present invention relates to a method for the treatment of strawberry gray mold pathogens ( Botrytis cinerea ) were isolated and their morphological and biochemical characteristics and sequence analysis (SEQ ID NO: 1). Streptomyces acidiscabies ). 1 and 3.

From the above results, the antimicrobially active strains of the present invention sorted and isolated were transformed into Streptomyces < RTI ID = 0.0 > acidisabies JA (II) -10, deposited at the Korean Agency for Agricultural Microbiology Resource Center, Rural Development Administration on September 17, 2009, and assigned the deposit number KACC 91492P.

The present invention relates to a method for producing Streptomyces < RTI ID = 0.0 > acid (II) -10 (KACC 91492P) strain or the strain Streptomyces < RTI ID = 0.0 > acidiscabies) JA (II) provides a microbial agent for plants gray mold having anti-fungal activity control containing cultures of strains -10 (KACC 91492P).

In the present invention, the microorganism preparation is a strain prepared by growing and growing Streptomyces acidis Jabs JA (II) -10 (KACC 91492P) in a medium and diluting it to 10 ⁵ cells / ml Characterized in that the plant gray mold disease is caused in strawberries.

More specifically, the supernatant of JA (II) -10 medium was prepared at each concentration (undiluted solution, 50%, 25, 12.5, 6.25, 0%) and then the gray mold growth The minimum concentration to inhibit was investigated. As a result, it was found that the gray mold growth pattern in the control and the gray mold growth pattern in the medium supplemented with the supernatant of JA (II) -10 were significantly different. Especially, the addition of the supernatant of JA (II) -10 % Of the medium, the growth of the gray mold was greatly suppressed. See FIG.

The present invention provides a method of controlling a plant ash fungus pathogen by treating a microorganism preparation according to the present invention, wherein the plant is a strawberry and is applied by a conventional method.

In detail, the microorganism preparation according to the present invention is applied to soil, crop leaves, stem, flower or fruit, and can be used by diluting a microorganism preparation in water. In addition, the microorganism strain of the present invention may be added with anti- It may further include a leaf surface spraying agent or a root spraying agent. In addition, pre-treatment of spraying before the onset and proliferation of gray mold can further increase the control effect.

The strain Streptomyces acidiscabies JA (II) -10 of the present invention has excellent antifungal activity against causative microorganisms of gray molds occurring in various crops, and in particular, It is possible to utilize it as an excellent and versatile biocide pesticide material which can solve the problem of ecosystem destruction and lethal toxicity by efficiently reducing mold disease.

The strain Streptomyces acidiscabies JA (II) -10 of the present invention has a large economic effect and high performance using microorganisms native to Korea, thereby increasing the commerciality and productivity of crops There is an effect that can be.

1 is a result of investigation of the antibacterial activity against Streptomyces acidis Jabs JA (II) -10 strain of Streptomyces sp . According to the present invention.
(A: untreated, B: JA (II) -10)
Fig. 2 is a graph showing the distribution of the strains of Streptomyces < RTI ID = 0.0 > acidiscabies ) JA (II) -10 in the culture medium.
(A: Nutrient Agar, B: Potato Dextrose Agar, C: Corn Meal Agar, D: Starch Casein Agar, E: Oat Meal Agar,
Figure 3 is a schematic diagram of the Streptomyces < RTI ID = 0.0 > acid < / RTI > species ) JA (II) -10,
Fig. 4 is a graph showing the distribution of Streptomyces < RTI ID = 0.0 > acidiscabies ) JA (II) -10,
FIG. 5 is a graph showing the distribution of Streptomyces < RTI ID = 0.0 > acidiscabies ) JA (II) -10,
FIG. 6 shows the results of investigating the availability of enzyme activity using Streptomyces acidiscabies JA (II) -10 strains of Streptomyces according to the present invention using API-ZYM,
FIG. 7 is a graph showing the distribution of Streptomyces < RTI ID = 0.0 > acidiscabies ) JA (II) -10,
(A: protease activity, B: chitinase activity)
FIG. 8 is a graph showing the distribution of Streptomyces < RTI ID = 0.0 > acidiscabies ) JA (II) -10 in the culture medium.
9 is a result of checking the effect of the Streptomyces acidiscabies JA (II) -10 strains on Staphylococcus aureus according to the present invention on a strawberry gray mold disease,
(A: untreated, B: JA (II) -10)
Fig. 10 shows the results of examining the effect of controlling Streptomyces acidiscabies JA (II) -10 strains of strawberry gray mold according to the present invention in a nursery seedling packaging.
(A: untreated, B: treated with JA (II) -10 strain, C:

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] Isolation and identification of a useful microorganism JA (II) -10 strain

Antimicrobial activity against strawberry gray mold pathogens ( Botrytis cinerea ) was selected by using 83 actinomycetes isolated from Saury litter and rhizosphere soil. The actinomycetes were first inoculated into selective medium (PDA medium), and after 24 hours, the strawberry gray mold pathogens were confluent and the activity was confirmed one month later. After the incubation, the growth inhibitor clear zone formed around the actinomycetes was measured to determine the inhibition rate against the gray mold pathogens ( Botrytis cinerea ).

As a result, 79 strains (96%) out of 83 strains isolated from leaves and rhizosphere of Sasa showed antimicrobial activity against strawberry gray mold pathogens and actinomycetes, which have antibacterial activity against gray mold pathogens, (II) -10, respectively. Among them, useful microorganism strains were selected and named as JA (II) -10.

After the test by the above-mentioned confluent culture method, And the inhibition rate was calculated. As a result, as shown in Fig. 1, when the selected JA (II) -10 actinomycetes were inoculated and the strawberry gray mold pathogens were replaced, after 1 week, the mycelium began to grow in the strawberry gray mold pathogens. JA (II) -10 strains showed slower growth of hyphae than control strains that were not cultured with actinomycetes. Thirty days after inoculation, the control cultivated mycelium throughout the petridish, while the strain JA (II) -10 , It was confirmed that the mycelium did not spread to the end of the petridish so that the antibacterial activity was 68.3%.

[Example 2] Useful microorganisms JA ( II ) -10 morphological biochemical characterization

1) Morphological characterization of JA (II) -10 strain

Yeast-extract malt-extract agar (International Streptomyces Project, ISP 2), Oatmeal agar (ISP 3) and Inorganic salts starch agar (ISP 3) were selected to examine the morphological characteristics of JA (Growth), sporulation, aerial mycelium after 15 days of culture on a Potato Dextrose Agar medium (ISP 4) medium, Nutrient agar (ISP 8), Corn Meal Agar (CMA) , Substrate mycelium and soluble pigment were prepared. ISP 2 (YMA medium) was prepared with 4 g of yeast, 10 g of malt extract and 4 g of glucose, ISP 3 (OA medium) was supplemented with 10 g of starch, 1.0 g of MgSO _{4 7} H ₂ O, 0.5 g of K ₂ HPO _{4 and} 10 g of Beef extract with ISP 8 (NA medium) with 20 g of Oatmeal and ISP 4 (SCA medium) , 10 g of peptone, and 5 g of sodium chloride. The PDA medium was adjusted to 200 g of potato, 20 g of glucose and 30 g of CA meal, While the one week incubation at 28 ℃ observed the group hyphae and aerial hyphae, the dye-forming ability.

As a result, as shown in FIG. 2, the strain JA (Ⅱ) -10 was found to be able to grow in yeast-extract malt-extract agar, Oatmeal agar, Inorganic salts starch agar, Nutrient agar, CMA and PDA medium I could.

In addition, in the above medium, both hyphae and spores were formed. Light gray with white, Nutrient agar medium in Yeast-extract malt-extract agar, Inorganic salts starch agar, Oatmeal agar, And white (white). The air hypha showed three colors such as brown, white and ivory, and it was confirmed that no pigment was formed in all media.

2) JA (II) -10 Molecular systematics  analysis

For the molecular genetic analysis of JA (Ⅱ) -10, DNA was extracted and polymerase chain reaction was used. 16S rDNA (about 1500 bp) base sequence was determined using ABI PRISM 310 Genetic Analyzer (Applied Biosystems) Homology was compared using the BLAST program of the DDBJ / NCBI / GenBank database. The homology of each nucleotide sequence was aligned in parallel using the alignment Clustal X algorithm, and the generation of the gene sequence was determined based on the local binding method

Analysis of the 16S rDNA sequence (1,224 bp) of JA (II) -10 revealed that the nucleotide sequence of FIG. 3 (SEQ ID NO: 1) was confirmed and the nucleotide sequence of the 16S rDNA base sequence of JA As a result of comparing the molecular systematic positions using the BLAST program of the RDP / NCBI / GenBank database, as shown in FIG. 4, it was confirmed that Streptomyces acidiscabies It was identified as Streptomyces acidiscabies with 99.7% homology with ATCC 49003 ^T D63865.

3) JA (Ⅱ) -10 Chemical classification of strains

For the chemical classification of JA (Ⅱ) -10, the microbial fatty acid composition of JA (Ⅱ) -10 was analyzed.

In order to investigate the cellular fatty acid composition of JA (II) -10, 50 ml of TSA (Trypticase Soy Broth Agar, Difco) medium was inoculated and cultured at 28 ° C for 5 days. The cells were collected by centrifugation at 8000 rpm for 10 minutes Respectively. After 50 mg (wet weight) of the collected cells was transferred to a teflon lined screw cap tube, the fructose fatty acid was converted to methyl ester by Ikemoyo & Miyagawa method and extracted. 50 mg of the cells were mixed with 1 ml of NaOH, and the mixture was incubated at 100 ° C. for 30 minutes to separate fructose fatty acids from the lipids of the cell membranes. Then, 2 ml of HCl was added thereto and the mixture was stirred at 80 ° C. for 10 minutes to form methyl esters .

1.25 ml of hexane-tert-butylmethylether was added and stirred for 10 minutes. Then, the supernatant was taken and the fatty acid was transferred from the receptive layer to the organic layer. The mixture was stirred for 10 minutes with 3 mL of NaOH and purified with saturated NaCl to remove foreign substances. 0.5 ml of the purified fatty acid was extracted and transferred to a GC vial. The fatty acid methyl ester was analyzed by Microbial Identification System (Midi; Microbial ID, Inc., Newark, U. S, A) (Stead, 1992).

As shown in Table 1, the major fatty acids were C16: 0 (14.78%), iso-C15: 0 (11.28%) and anteiso-C15: 0 %), And it was confirmed that various branched fatty acids such as 13: 0 iso, 14: 0 iso, 15: 0 iso, 15: 0 anteiso, 16: 0 iso, 17: 0 iso and 17: 0 anteiso there was. These results were consistent with the major fatty acid composition of actinomycetes, gram - positive bacteria.

3) Quinone species analysis of JA (Ⅱ) -10 strain

Identification of isoprenoid quinone species was carried out by using a freeze dryer (Eyela, Japan) after collecting the cells cultured in SCA medium for 2 weeks using the method of Collin and Jones. 20 ml of a chloroform: methanol (2: 1) solution was added to 400 mg of dried cells, and the mixture was treated for 24 hours to extract isoprenoidquinone. The cell components in the extract were filtered using a filter paper, and the filtrate was concentrated on a vacuum rotary evaporator (Eyela, Japan) for 30 minutes. 4 ml of acetone was added to the evaporator flask, and the isoprenoid quinone was dissolved and concentrated three times with pure nitrogen (N ₂ ) gas. The isoprenoidquinone concentrate was spotted on a TLC (Merck Kiesel-gel 60 F254, 20x20, 0.5 mm thickness) plate with ubiquinone and menaquinone standards. 100 ml of petroleum benzene / diethyl ether (9: 1) developing solution was added to the developing tank, and the developing solution was developed to the extent of about 3 cm from the top of the TLC plate and confirmed at UV 254 nm . Each quinone band was scraped off with a razor, transferred to tube e, dissolved in 1 ml of acetone, and filtered through a 0.42 ㎛ syringe filter. The filtered acetone extract was concentrated with nitrogen (N ₂ ) gas. Analysis of quinone species was performed with HPLC (Shimadzu, Japan) using a 5C18 column (4.6 x 150 mm; Nacalai Tesqu) with standard materials (Collins, MD and D. Jones., 1981).

As a result, as can be seen from the results of Table 2 and FIG. 5, the quinone species of JA (II) -10 was developed by TLC, and then, in the UV 254 nm, (H4), MK-9 (H6) and MK-9 (H8) were found to contain 3.79%, 54.58% and 40.3%, respectively, as determined by HPLC analysis .

5) JA ( II ) -10 physiological characteristics

To investigate the enzyme activity of JA (II) -10, Zym was analyzed using API ZYM kit. A single colony of JA (II) -10 was suspended in distilled water, inoculated on a cube of a strip maintained in water, and then cultured in a dark state at 37 ° C for 4 hours. After 4 hours, ZYM A and ZYM B reagents were inoculated one by one into each cuplet. After 5 minutes, the reaction results were read as - / +.

As a result, there was a positive reaction with Esterase, Esterase Lipase, Leucine arylamidase, Valine arylamidase, Crystine arylamidase, Trypsin, α-chymotrypsin, Acid phospatase and Naphtol-AS-BI-phosphohydrolase.

6) Enzymatic characterization of JA (II) -10 strain

end. Protein Enzyme Activity Assay

Proteolytic enzyme activity assays were performed by adding 1% skim milk to a nutrient agar medium (Nutrient Broth, NB) diluted 1/10 and using a pure JA (II) -10 strain , And then cultured at 28 ° C for 7 days to investigate the protease production ability through the cleavage zone formed on the solid medium.

As a result of culturing for 1 week on a skim milk agar containing a non-fat milk, it was confirmed that a proteolytic enzyme was formed by forming a substrate decomposition band having a radius of 1.5 cm as shown in FIG. 7A.

I. Chitinase activity assay

To investigate chitin degradation, a nutrient agar medium (NB) diluted 1/10 was added to 1% colloidal chitin. The chitin resolving ability was determined by inoculating JA (II) -10, which was purely isolated, into the above plate medium, followed by culturing at 28 ° C for 7 days, and cleaving the chitin through the substrate cleavage zone formed on the solid medium.

As shown in FIG. 7 (B), chitin degradation activity was observed in a plate medium supplemented with 1% colloidal chitin. However, the JA (II) -10 strain was found to have an enzyme that decomposes chitin It was confirmed that it was generated,

[Experimental Example 1] Minimum concentration of antimicrobial activity of strain Staphyllacesis sydischevis JA (II) -10

The supernatant of JA (II) -10 medium was prepared for each concentration, and then added to PDA (Difco Potato Dextrose Agar) medium and intermediate layer medium to prepare the medium. PDA medium was prepared by using Potato Dextrose Agar medium prepared by Difco. The middle layer medium was prepared by preparing the supernatant of JA (II) -10 medium by concentration (undiluted solution, 50%, 25, 12.5, 6.25, 0% Lt; / RTI > medium. The cultured gray mold hyphae were scraped with a spreader and suspended in 10 ml of sterilized water. 100 .mu.l of each medium was inoculated into the medium of each concentration to determine the minimum concentration that inhibited gray mold development after 10 days.

As a result, as shown in FIG. 8, it was found that the gray mold growth pattern in the control and the gray mold growth pattern in the culture medium containing the supernatant of JA (II) -10 were significantly different, and JA (II) The growth of gray mold was significantly inhibited in medium containing more than 50% of the supernatant of 10 strains. It was confirmed that the minimum concentration of gray mold inhibition by the addition of the supernatant of JA (II) -10 was 50%.

[Experimental Example 2] Effect of Staphylococcus aureus Siebische JA (II) -10 on the control of gray mold disease

1) Evaluation of the efficacy of JA (II) -10 in the cabin

In order to investigate the antagonistic effect of JA (II) -10 on the gray mold of strawberry fruit in the test package, eco-friendly strawberries were distributed from the Nonsan strawberry test site without treatment of the gray mold control agent and used as the test material .

The pre-packaged strawberries were placed in a transparent case prepared by repeating the procedure in three replications of 10 without rinsing to induce spontaneous generation of gray mold. The control was spray-treated with sterilized water. The JA (II) -10 cells were sprayed homogeneously and homogeneously without lumping. Each treatment was incubated for one week in a culture room with a temperature of 23 ° C, and the efficacy was verified by counting the gray mold infected with the gray mold according to each treatment.

As shown in FIG. 9, the disease began to occur from about 3 days after inoculation and severe symptoms were observed from about 5 days after inoculation. JA (II ) -10 bacterium showed a significantly decreased incidence of gray mold compared to the control.

2) JA (II) -10 strain packaging test

In order to verify the effect of the JA (II) -10 strain of the present invention on the control of the strawberry gray mold, a packaging demonstration test was conducted. The test was carried out at 30 weeks of treatment in the Nonsan strawberry test center of Chungnam Agricultural Research and Extension Service.

In the gray mold infecting strain, 4 plates with hyphae spread widely in the PDA medium were prepared, and a certain amount of distilled water was added thereto, and only the hyphae part was scraped with a spreader carefully. After that, the cells were adjusted to PDB solution so that the inoculation concentration was 10 ⁵ cells / ml and used as an inoculation source for the occurrence of gray mold infestation.

In order to inoculate the prepared strawberry plant, it is necessary to spray the seedlings of the strawberry at the flowering part and the fruit part so that the inoculation liquid flows down sufficiently, and then keep the packaging environment so as to smoothly induce the occurrence of the gray mold gave.

The infection was confirmed one week after the gray mold infestation. In general, it is known to infiltrate into plants 2 days after inoculation.

As a result of the investigation of the gray mold infections after one week in the present experiment, the infected seedlings showed spotting symptoms on the flowering part and the occurrence of the insect was found to have occurred.

Based on the above preliminary tests, 30 treatments of strawberry per plant were treated with 3 treatments (control) (sterilized water), JA (II) -10 and control chemicals (Ecozet) After three inoculations, the control of gray mold infection was examined.

The strain JA (II) -10 was cultured in a 5000 ml Erlenmeyer flask containing 1000 ml of SCB medium and cultured for 2 days in a 10 ml SCB medium. Cells were collected by collecting the cells of the cultured culture for one week, and then washed twice with sterilized water to remove the medium components. To remove the agglomerated fungi, they were crushed and mixed well and treated with an artificially infested strawberry plant. The control drug (Ecozet) was diluted to 500 times and treated in the same manner.

The prepared strawberry seedlings were divided into one treatment group and placed in the control (sterilized water), control drug treatment group and JA (II) -10 treatment group from the front, and each treatment solution was inoculated three times at weekly intervals , And the area of disease diffusion (AUDPC).

As a result, as shown in FIG. 10 and Table 4, the inhibitory effect of the strawberry gray mold disease on the antimicrobial active JA (II) -10 strain of the present invention was 19.9% in the control, whereas JA (II) -10 The rate of disease progression was 8.3% in the strain treated, and it was confirmed that it was statistically significant.

In contrast, 33.3 of the control agent showed a good control effect as compared with 33.3 of the control agent. In the bottle diffusion area survey (AUDPC), JA (II) -10 strains showed excellent inhibitory effect against gray mold.

Agricultural Biotechnology Research Institute KACC91492 20090917

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Claims (7)

delete delete A culture of Streptomyces acidiscabies JA (II) -10 (KACC 91492P) strain or Streptomyces acidiscabies JA (II) -10 (KACC 91492P) ( Botrytis cinerea ), a microorganism preparation for controlling a strawberry gray mold disease having antifungal activity against Botrytis cinerea of a strawberry gray mold disease. The method of claim 3,
The microorganism preparation is produced by cultivating Streptomyces acidiscabies JA (II) -10 (KACC 91492P) strain in a medium at a concentration of 10 ⁵ cells / ml. Microorganism preparation for controlling strawberry gray mold disease with activity. delete delete delete

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