KR101060573B1 - Asp-p 1441 of the genus Streptomyces of the new strain and plant disease control agents using the same - Google Patents

Abstract

The present invention relates to a new strain Streptomyces sp. AG-P 1441 (KCTC 11603BP) and a plant disease control agent using the same, specifically, an improved strain from Streptomyces sp. AG-P (KCTC 8965P) strain The novel strain Streptomyces genus AG-P 1441 (KCTC 11603BP) produces paromomycin, Phytophthora sp., A plant late blight , and Pythium sp. By confirming selective antimicrobial activity against phytopathogenic fungi (Oomycete), such as bacteria, it can be usefully used as a biocontrol agent for the control of plant diseases such as plant late blight and wearing disease using the strain. have.

Streptomyces sp. AG-P 1441 (KCTC 11603BP), paromomycin, late blight, incoming germs, biopesticides.

Description

A new Streptomyces sp. AG-P 1441, and an agent for control of plant diseases using the same

The present invention relates to a novel improved strain and a plant disease control agent using the same.

Plant diseases caused by the late blight Phytophthora and the stock bacteria Pisium are very numerous and the damage is enormous. Around 60 kinds of late blight and more than 1,000 late blight host plants have been reported all over the world. In Korea, 16 kinds of phytophthora are known to cause 46 crops. For example, Phytophthora infestans are primarily in potatoes and tomatoes, and Phytophthora capsici occurs in all plant species, including peppers, watermelons, cucumbers, melons, and tomatoes. It is known that one strain species occurs in many hosts. In particular, the phytophthora infestans and phytophthora capsaish are causing great damage to potatoes, tomatoes and peppers in Korea.

Plague germs overwinter in diseased plants in the soil in mycelial state, and after the growth of pathogens, they appear on the ground and become secondary infectious agents. Plague is one of the most important plant diseases that causes a lot of losses in crop production because the disease develops in a cool and humid environment. Rice seed rot and stocking disease caused by the pathogens in Pisium are well known all over the world, including our country, and the damage is serious. In Korea, Psidium spp. Is known as a major pathogen that causes root rot, including seedling rot of various crops such as tomatoes and cucumbers, and ginger root rot.

On the other hand, late blight, a type of oomycetes, is a typical soil pathogen that has been included in the fungal taxonomy so far, but has recently been controlled by the International Society of Fungi as classified as a protist. The fungicides used for this purpose also differ in their properties, and metalaxyl, dimethomorph, and focetyl-A1, which are different from the fungal fungicides which are generally used, are used.

However, these are chemical synthetic pesticides, and very few microbial fungicides which directly use microbial strains have been developed. In addition, many crops are hydroponically grown by genetic engineering techniques during agricultural production. However, when hydroponic farming is carried out, damage caused by late blight and incoming pathogens, which prefer water, has been reported, and most existing pesticides are insoluble in water. It is reported that the control rate is very weak or ineffective.

Therefore, as the interest in environmentally friendly agriculture grows, the interest in controlling methods using biopesticides using microorganisms is increasing. Therefore, development of microbial fungicides using microbial strains or microbial cultures itself is an urgent task.

Therefore, the inventors of the present invention, while using the soil microorganism actinomycetes, while researching and developing a method for controlling the late blight bacteria or incoming bacteria belonging to the biological, from the existing strain of Streptomyces genus AG-P (KCTC 8965P) Improved novel strain of Streptomyces genus AG-P 1441 (KCTC 11603BP) produces paromomycin and exhibits strong antibacterial activity against Phytophthora sp. By confirming this, the present invention was completed by confirming the development potential as a plant disease control agent such as late blight and wearing disease using the strain.

An object of the present invention is to provide an improved novel strain Streptomyces genus AG-P 1441 strain (KCTC 11603BP).

Still another object of the present invention is to provide an environment-friendly microbial agent effective for controlling plant diseases such as late blight and wearing disease by using the strain.

In order to achieve the above object, the present invention provides a Streptomyces sp. AG-P 1441 strain (KCTC 11603BP) characterized by having phytopathogenic fungi and phytopathogen control activity. .

The present invention also provides a plant disease control agent comprising the strain or its culture as an active ingredient.

In addition, the present invention provides a plant disease control method comprising the step of treating the strain, its culture solution or the plant disease control agent to soil, plants or seeds of plants.

Streptomyces sp. AG-P 1441 (KCTC 11603BP) strains improved from Streptomyces sp. AG-P (KCTC 8965P) strains of the present invention are compared to the AG-P parent strain (KCTC 8965P), paromomycin. Produces significantly more than 20 times, and control by using the strain by confirming that it exhibits selective antimicrobial activity against phytopathogenic fungi (Oomycete), such as Phytophthora sp. It can be usefully used as a biocontrol agent for the control of plant diseases such as plant late blight and wearing disease which are known to be difficult.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Streptomyces sp. AG-P 1441 strain (KCTC 11603BP).

The strain may produce a large amount of paromomycin, and may have activity in controlling plant late blight and respiratory disease caused by phytopathogenic fungi.

In the present invention, in order to search for new microorganisms for controlling plant diseases, paromomycin among the mutants modified by UV irradiation from the existing strain of Streptomyces genus AG-P (KCTC 8965P) was significantly higher than the parent strain. Mutant strains were selected (see Table 1).

In the present invention, to identify the mutant strains, the morphological, cultural, physiological characteristics, and sugar availability of the strains were analyzed in comparison with the parent strains.

Specifically, the morphological characteristics of the variant strain of the present invention is that the chain form of spores are spiral, the number of spores per chain is 10 to 20, the spores are elliptical in size 0.5 ~ 0.8 × 0.6 ~ 1.0 ㎛, which is a parent strain There was a difference in the shape and size of the spores (see Table 2, Figures 2 and 3).

In addition, the cultivation characteristics of the mutant strains of the present invention showed good growth in yeast, malt agar, oatmeal agar, inorganic salt, starch agar, peptone, yeast, iron agar, tyrosine agar, and Bennett agar, while glycerol and asparagine agar. The average level was shown in, and the color of star mycelia and matrix mycelium was different from the parent strain in a specific medium (see Table 3 and Table 4).

In addition, the physiological characteristics of the mutant strains of the present invention showed melanin pigment formation ability was negative, diaminopimellic acid showed LL type, and skim milk powder hydrolysis ability was positive, similar to the parent strain. See Table 5).

In addition, the carbon availability of the mutant strains of the present invention is L-arabinose, D-mannose, D-sorbitol, D-xylose, L-asparagine, D-aspartic acid, sucrose, β-methyl-D-glucoside, D-cellobiose, methyl-pyruvic acid, glycyl-L-proline, pyruvic acid, D-galacturonic acid, and the like were used well, and it was confirmed that there was a large difference between the parent strain and the carbon availability (Table 6). Reference).

In addition, the paromomycin activity of the mutant strains of the present invention was compared with that of the parent strain. As a result, the parent strain exhibited 20 ppm concentration activity of paromomycin, whereas the mutant strain exhibited 400 ppm concentration activity of paromomycin, indicating that the activity was 20 times higher than that of the parent strain (see FIG. 3).

As a result of examining the morphological, cultural, physiological characteristics and sugar availability of the mutant strains of the present invention, the strains were identified as novel strains having different characteristics from the parent strain. It was deposited on November 23, 2009 with the Korea Institute of Biotechnology Gene Bank (KCTC) and was given accession number KCTC 11603BP.

In addition, the present invention provides a plant disease control agent comprising a strain of Streptomyces genus AG-P 1441 of the present invention or a culture solution of the strain as an active ingredient.

The plant disease control agent may further include an agriculturally acceptable carrier in addition to the strain or its culture.

In the present invention, in order to determine the antimicrobial activity of the strain Streptomyces genus AG-P 1441 of the present invention, the antimicrobial activity against the late blight bacteria was assayed using the culture medium of the strain. As a result, the strain exhibited a strong antimicrobial activity against the phytophthora capcisi , a late blight bacterium , and showed significantly superior antimicrobial activity compared to the parent strain (see FIG. 4).

In the present invention, in order to determine the antimicrobial activity of the Streptomyces sp. AG-P 1441 strain of the present invention in vivo , it was directly treated to plants and assayed in the greenhouse. As a result, there was a control effect against rice fever, rice blast disease, tomato late blight, wheat red rust, etc., especially showed a strong control effect against tomato late blight and wheat red rust (see Table 7).

Therefore, the strain Streptomyces genus AG-P 1441 of the present invention produces significantly more than 20 times more paromomycin than the parent strain, and against phytopathogenic fungi (Oomycete), such as plant late blight and stock bacteria By confirming the selective antimicrobial activity, it can be seen that it can be usefully used as a biocontrol agent (biocontrol agent) for the control of plant diseases, such as plant late blight and wearing diseases known to be difficult to control using the strain. .

In addition, the present invention provides a plant disease control method comprising the step of treating the strain of Streptomyces genus AG-P 1441, the culture solution of the strain, or the plant disease control agent of the present invention to the soil, plant or plant seeds. .

The strain, its culture solution or a plant disease control agent containing them may be sprayed on the soil around the crop in a solid state, irrigation to the plant in a liquid state, immersed or sprayed on the seeds of the crop or coated on the seed, but is not limited thereto. Do not.

In the case of the immersion method, the strain, its culture solution or a plant disease control agent containing them can be poured into the soil around the plant or the seeds can be immersed in the culture solution and the preparation, and when sprayed, it is well known in the art. The technique can be applied to flow through the plant, but is not limited thereto.

The plant may be a vegetable crop, preferably one selected from tomatoes, cabbages, lettuce, cucumbers, peppers, green onions and garland chrysanthemum, but is not limited thereto.

AG-P 1441 strain of the genus Streptomyces of the present invention produces significantly more than 20 times more paromomycin than the parent strain, and strong antibacterial against phytopathogenic fungi (Oomycete), such as plant late blight and stock bacteria By showing activity, it can be seen that it can be usefully used for the control of plant diseases such as plant late blight and wearing disease.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Improvement of Strains

The present inventors incubated the Streptomyces genus AG-P strain (Accession No .: KCTC 8965P) in Bennett's agar plate medium at 28 ° C. for 7 days, and then used a glass fiber using a syringe (syringe) of the parent strain. The cells were collected by filtration and diluted in 50 mM Tris / malate buffer (pH 8) to a concentration of 10 ⁶ to 10 ⁸ spores ml / L. Subsequently, after spreading on Bennett's agar plate medium, UV (253.7 nm / 30 W) was irradiated for 10-30 seconds at a distance of 30 cm. After UV irradiation, the Bacillus subtilis ATCC 6633 assay cultured at 10 ⁶ cells ml / L concentration was applied to LB agar medium, and then hardened. Variants were selected according to the size of the inhibition zone which appeared after incubation at 37 ° C. After application of Bacillus strain after UV irradiation, it was allowed to stand for 1 hour at 4 ℃ to ensure the stability of the mutant strains.

As a result, it can be seen that the improved strain 'strain Streptomyces genus AG-P 1441' produces higher paromomycin (paromomycin) than the parent strain Streptomyces genus AG-P (Table 1, 1 and 3).

Inhibitory Activity of Parental and Mutant strains and Comparison with Paromomycin Strains Inhibitory ring diameter (mm) Streptomyces sp. AG-P 10.0 (20 ppm) Streptomyces sp. AG-P 1441 21.0 (400 ppm) Paromomycin 20 ppm 10.0 Paromomycin 50 ppm 12.5 Paromomycin 100 ppm 14.5 Paromomycin 200 ppm 17.0 Paromomycin 400 ppm 21.0 Paromomycin 500 ppm 23.0

Example 2 Characterization of AG-P 1441 of the Genus Streptomyces spp.

To identify the strains, the inventors analyzed the various characteristics of the strains with the parent strains.

<2-1> Morphological characteristics of bacteria

In order to analyze the morphological characteristics of the strain, according to the International Streptomycets Project (ISP) regulations were incubated for 14 days at 28 ℃, and the shape was examined under an optical microscope and an electron microscope.

As a result, the Streptomyces genus AG-P 1441 strain showed a spiral form of spores, the number of spores per chain was 10-50, the surface of the spores was smooth, the spores were elliptical and 0.5-0.8 in size. It was x 0.8-1.2 micrometer (Table 2, FIG. 2, FIG. 3).

Comparison of Morphological Characteristics of Parent and Mutant strains characteristic Streptomyces sp. AG-P Streptomyces sp. AG-P 1441 Spore form Cylindrical Oval Spore Size (μm) 0.8 to 1.0-1.0 to 1.4 0.5 to 0.8-0.8 to 1.2 Spore surface Smoothness Smoothness Spore chain mode gyre Compact spiral Spores Per Chain 20-50 10 to 50 Spore motility Non-mobility Non-mobility

<2-2> Culture characteristics of bacteria

In order to analyze the culture characteristics according to the various media of the strain, after culturing for 14 days at 28 ℃ under various ISP medium conditions, the culture physiological characteristics of the strain were investigated.

As a result, the growth of AG-P 1441 strain of Streptomyces showed relatively good growth in yeast, malt agar, oatmeal agar, mineral salt, starch agar, peptone, yeast, iron agar, tyrosine agar, and Bennett agar. And was common in glycerol and asparagine agar. On the other hand, the specific mycelia of the mycelia and substrate mycelia were different from the parent strain (Table 3 and Table 4).

Culture Characteristics of AG-P Strains of Streptomyces spp. badge growth Mycelia
color Substrate hyphae
color receptivity
Pigment Yeast extract-malt extract agar (ISP No. 2) Good Gray Pale brown - Oatmeal Baby (ISP No. 3) Good Gray Pale yellow - Inorganic salt-starch agar (ISP No. 4) Good Gray Pale yellow - Glycerol-asparagine agar (ISP No. 5) Moderato Pale gray Pale brown - Peptone-Yeast Extract-Iron Agar (ISP No. 6) Good Pale gray Brown - Tyrosine Baby (ISP No. 7) Good Gray Pale brown - Bennet Baby Good Gray Pale brown -

Culture Characteristics of Streptomyces sp. AG-P 1441 Strain badge growth Mycelia
color Substrate hyphae
color receptivity
Pigment Yeast extract-malt extract agar (ISP No. 2) Good Gray Pale brown - Oatmeal Baby (ISP No. 3) Good Gray Pale yellow - Inorganic salt-starch agar (ISP No. 4) Good Gray Pale yellow - Glycerol-asparagine agar (ISP No. 5) Moderato Pale gray Pale yellow - Peptone-Yeast Extract-Iron Agar (ISP No. 6) Good Pale yellow Pale brown - Tyrosine Baby (ISP No. 7) Good Pale gray Pale brown - Bennet Baby Good Gray Pale brown -

<2-3> Physiological characteristics of bacteria

In order to analyze the physiological characteristics of the strain, after incubation for 14 days at 28 ℃ using Schilling-Gurtrib agar medium, melanin production capacity, diamino pimeline acid production capacity and skim milk powder hydrolysis capacity was investigated.

As a result, the AG-P 1441 strain of Streptomyces genus showed negative melanin-forming ability, the diaminopimellic acid showed LL type, and the skim milk hydrolysis capacity was positive as the parent strain. Is shown (Table 5).

Comparison of Physiological Characteristics of Parent and Mutant strains Survey Item Streptomyces sp. AG-P Streptomyces sp. AG-P 1441 Melanin Pigment Generation voice voice Diaminopimeline acid production LL LL Skim milk powder hydrolysis positivity positivity

<2-4> Characteristics of Bacterial Carbon Sources

In order to investigate the carbon availability of the strain, using a Schilling-Gutribe agar medium for 14 days at 28 ℃ was investigated the carbon source availability.

As a result, the strain AG-P 1441 of Streptomyces genus, unlike the parent strain, had L-arabinose, D-mannose, D-sorbitol, D-xylose, L-asparagine, D-aspartic acid, sucrose, β -Methyl-D-glucoside, D-cellobiose, methyl-pyruvic acid, glycyl-L-proline, pyruvic acid, D-galacturonic acid and the like were used well.

Comparison of Carbon Source Availability in Mother and Variants Party Usability Streptomyces sp. AG-P Streptomyces sp. AG-P 1441 L-Arabinose - + Succinic acid + - D-Galactose + + D-Trehalose + + D-Mannose - + D-Sorbitol - + Glycerol + + D-Xylose - + D-Mannitol + + D, L-Malic Acid + - D-Ribose + + L-Rhamnose - - D-Fructose + + Acetic acid + - α-D-Glucose + + Maltose - - D-Melibiose - - L-Asparagine - + D-Aspartic Acid - + α-D-Lactose - - Sucrose - + β-Methyl-DGlucoside - + Adonitol + - Maltotriose + + 2-Deoxy Adenosine + - M-Inositol + + Propionic acid + - D-Cellobiose - + Inosine + - Methyl pyruvate - + L-Malic Acid + + Glycyl-L-proline - + Pyruvic acid - + D-Galacturonic Acid - +

As described above, as a result of investigating the morphological, culture, physiological characteristics and sugar availability of the strain Streptomyces genus AG-P 1441 of the present invention, it was identified as a novel strain, the strain 'Streptomyces genus AG-P 1441 strain, and was deposited with the Biotechnology Research Institute Gene Bank (KCTC) on November 23, 2009 and was assigned accession number KCTC 11603BP.

Example 2 of Streptomyces genus AG-P 1441 in vitro  Antimicrobial activity

The present inventors assayed the antimicrobial activity against the late blight bacteria by using the culture medium of the Streptomyces sp. AG-P 1441 strain obtained in <Example 1> in order to determine the antibacterial activity of the strain Streptomyces genus AG-P 1441. It was. Specifically, antimicrobial activity was investigated in vitro using Phytophthora capcisi as a test strain. Potato liquid medium was treated with AG-P 1441 strain culture medium of Streptomyces genus to 10% concentration in V8 juice medium to observe the growth of bacteria.

As a result, the strain AG-P 1441 of the genus Streptomyces showed very strong antibacterial activity against the phytophthora capcisi , a late blight bacterium , which was remarkably higher than that of the parent strain Streptomyces genus AG-P. It was confirmed that the high antibacterial activity (Fig. 4).

Example 3 of Streptomyces genus AG-P 1441 in vivo Antimicrobial activity

The inventors of the present invention have directly tested the activity of a strain of AG-P 1441 of Streptomyces genus in vivo against tomato late blight and several other plant diseases and assayed its activity in a greenhouse.

As a result, there was a control effect against rice fever, rice blast disease, tomato late blight, wheat red rust, etc., in particular against tomato late blight and wheat red rust disease (Table 7).

In vivo Control Effects of Various Plant Pathogens Plant pathogen Bottle Control Rate (%) Pyricularia oryzae 80 Rice wearing bottle ( Rhizoctonia solani ) 70 Cucumber gray mold disease ( Botrytis cinerea ) 50 Tomato Plague ( Phytophthora infestans ) 100 Wheat Red Rust ( Puccinia recondita ) 100 Barley powdery mildew ( Erysiphe graminis f . Sp. Hordei ) 65

As a screening method for investigating the control effect against tomato late blight , spore concentrations of Phytophthora infestans were made at 10 ⁵ sporangia / ml, and cold-treated at 13 ° C. for 2.5 hours. After the distillation, 3 ml of Streptomyces genus AG-P 1441 strain culture solution was spray-inoculated onto the treated tomato seedlings (second to third leaf). Tomato seedlings were treated for 48 hours in a 20 ° C. wet room and then incubated for 3 days in a 20 ° C. constant temperature and humidity room (relative humidity above 95%), and used for the experiment. The control effect on other rice fever, rice blast disease, cucumber ash fungus, wheat red rust disease, and barley flour disease was tested in a similar manner.

As a result, the control effect against tomato late blight was very high, and this result indicated that the phytophthora infestans, the tomato late blight bacterium, was as selective as the activity in the medium. In addition, the results showed that the inhibitory and control effects were very high also against the strains of the phytophthora, which is a late blight bacillus, and the strain of Pisium, which is a similar fungus, and could be effectively used to control rice wearing diseases.

As described above, the novel Streptomyces sp. AG-P 1441 strain of the present invention can be usefully used in the development and production of biocontrol agents (biocontrol agent) for controlling plant diseases in an environmentally friendly and economical manner.

1 is a diagram showing the morphological characteristics on the Bennett's agar medium of the strain strain Streptomyces genus AG-P (KCTC 8965P) and the strain strain Streptomyces genus AG-P 1441.

2 is a diagram showing spore photographs (SEM) of the strain strain Streptomyces genus AG-P (KCTC 8965P) and the strain strain Streptomyces genus AG-P 1441.

3 is a diagram showing the paromomycin activity of the strain strain Streptomyces genus AG-P (KCTC 8965P) and the strain strain Streptomyces genus AG-P 1441.

Figure 4 is a diagram showing the antimicrobial activity against Phytophthora capcisi of the strain strain Streptomyces genus AG-P (KCTC 8965P) strain and strain strain Streptomyces genus AG-P 1441.

Claims (7)

Streptomyces sp. AG-P 1441 strain (KCTC 11603BP), characterized by having phytopathogenic fungi and plant diseases caused by fungi. Plant disease control agent comprising the strain of claim 1 or a culture thereof as an active ingredient. The plant disease control agent according to claim 2, further comprising an agriculturally acceptable carrier. The plant disease control agent according to claim 2, wherein the plant disease is plant late blight or wearing disease. A method of controlling plant diseases comprising treating the strain of claim 1, its culture solution or the plant disease control agent of claim 2 to soil, a plant or a seed of a plant. The method of controlling plant diseases according to claim 5, wherein the plant is irrigated, sprayed or sprayed on the seeds of the crop in a liquid state, or coated on the seeds. The method of claim 5, wherein the plant disease is plant disease or wearing disease.

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