KR100397796B1 - Antifungal Biocontrol Agents and Use thereof - Google Patents

Abstract

The present invention relates to an antimicrobial microbial agent, a manufacturing method, a treatment method and its use which can enhance the growth of a plant by controlling fungal pathogens of the plant. Antimicrobial microbial agents of the present invention comprise at least one novel antimicrobial microbial strain of Streptomyces spp . WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0342BP), and the viability and activity of these strains It contains a microbial delivery medium capable of maintaining a stable state.

The antimicrobial microbial agent of the present invention is a control of fungal pathogens, including lysatonia, phytophora, sclerotenia, piculeria, choletotricum, botrytis, spheroceca, and the like, which cause diseases of various plants. It is highly active and can be treated in many ways in plants, especially cucumbers, peppers, grasses, pumpkins, strawberries, melons, lettuces, roses, tomatoes, leeks, green onions, tobacco, rice, etc., mosslock, brown patches, large patches, It can be effectively used to control a variety of seeding and polio diseases, including plague, powdery mildew, gray mold, anthrax, leaf blight, blast, and dollar spot disease.

Description

Antimicrobial microbial agent and its use {Antifungal Biocontrol Agents and Use}

The present invention relates to a microbial agent, a manufacturing method, a treatment method, and a use thereof having antimicrobial properties, and more particularly, plant growth by controlling fungal pathogens that cause disease in plants by growing in the root zone of soil where plants are growing. It relates to an antimicrobial agent, a manufacturing method, a treatment method and its use that can enhance the.

Fungal pathogens in the soil cause significant economic losses to agriculture and horticulture. Representative diseases that cause enormous economic losses to agriculture and horticulture include seed rot, root rot, mozambique, psium blight, brown patches, large patches, dallas spot disease, anthrax, powdery mildew, gray mold, paperback disease and blasting disease. Can be mentioned. Powdery mildew and ash mildew disease, which causes folliar diseases, develops on a wide range of plants, and has a wide host range, rapid disease rate, and strong pathogenicity. In particular, Rhizoctonia solani is very pathogenic and seedling diseases such as seed rot, root rot and mosslock disease on a wide range of host plants and polyfolia on leaves, petioles and stems. diseases, and the economic loss from the damage is very large. As an example, Raiztonia Solani AG 1 (IB) causes Raiztonia brown patches on bentgrass, green grass of golf courses, as well as general crops including cucumbers, peppers, etc. 2 (IV) causes a large patch on the golf course fairway grass, and the damage area and the degree of damage are enormous. In addition, Phytophthora capsici is a causative agent of cayenne pepper disease and is a very pathogenic pathogen. Once infected by this strain, it causes a fatal loss that cannot prevent the development of cayenne pepper disease. It occurs on all the above-ground vegetation at the time of high temperature and humidity, and it causes fatal economic loss to growers by killing the plants. Like this, Raiztonia solani and phytosov capsaisi are highly pathogenic, form mycelium or spores and linger for a long time even under harsh environmental conditions, and then the disease recurs when the disease occurs. do. Therefore, although compound fungicides are regularly used to control diseases of plants caused by Raiztonia solani and phytopusora capsaish, the emergence of drug-resistant strains due to the intensive use of fungicides and In the case of massive growth and perennial turf, the latent resistance of resistant pathogens, the effective control of pepper blight and brown and large patches of turf has become more difficult.

Repetitive use of chemical synthetic pesticides not only causes residual toxicity and environmental pollution, but also sprays on crops that have already grown, even if the pesticides are sprayed. You have no control Therefore, in order to continuously and effectively control the diseases of pepper crops caused by phytoplasma capsaici and general crops and grasses caused by Laytononia solani, microorganisms, in particular, specific microorganisms that grow in the root zone, are used to sustain pathogens. It is reasonable, economical, and effective in terms of environmental protection by controlling and controlling the onset of plants by controlling them. Recently, research and development of microbial fertilizers, microbial preparations, and microbial pesticides using microorganisms have been conducted.

As a related art in the related art, EP Publication No. 0 294 053 discloses a composition comprising an N-acyl lactam compound and a microbial strain as a composition for promoting plant growth, and US Pat. No. 4,595,589 discloses peat. A method for promoting plant growth and preventing diseases using a microbial delivery medium composition in a suspension state containing the composition and a microbial strain is disclosed. US Pat. No. 5,403,584 discloses peat moss and sand. And a method comprising a composition comprising a microbial delivery medium and a microbial strain as a main component and corn for the above uses is disclosed. The EP patent still suffers from the use of chemically synthesized compounds, and since the compositions disclosed in the above US patents are all made of materials obtained from nature, it helps to solve the environmental pollution problem. Due to component problems, it is not easy to be prepared in powder form after sterilization, and it is difficult to remain stable even in suspension form, so it is not very effective in the case of treating directly to the seed of the plant. In addition, in a microbial preparation including a microbial strain having antimicrobial properties, the antimicrobial activity of the antimicrobial microbial strain, which is the most important factor, must be higher than a certain level and the activity must be stable, but it is not satisfactory in the microbial preparations up to now. No results were possible.

Therefore, the technical problem to be achieved by the present invention is to isolate the antimicrobial microbial agent comprising a novel microbial strain having a protective activity against plant diseases, in particular plant diseases caused by Laytononia Solani and phytosov capsaici. To provide.

Another technical problem to be achieved by the present invention is to provide a method for producing an antimicrobial microbial agent, which is suitable for plant seed, root or soil treatment as a method for producing a microbial agent as described above and suitable for maintaining the antimicrobial microbial strain in a viable state. .

Another technical problem to be achieved by the present invention is to provide a method for easily treating the antimicrobial microbial agent.

Another technical problem to be achieved by the present invention is to provide a use for the use of the antimicrobial microbial agent to prevent various plant diseases.

In order to achieve the above technical problem, in the present invention, in the antimicrobial microbial agent that can improve the growth of plants by controlling fungal pathogens of plants, Streptomyces spp . (WC20 03KBP) and Streptomyces spp . Provided is an antimicrobial microbial agent comprising at least one antimicrobial microbial strain selected from the group consisting of WYE 324 (KCTC 0342BP).

Here, the plant is not particularly limited, but is preferably a field crop, a rice crop, a horticulture and a flower crop, particularly cucumbers, peppers, pumpkins, strawberries, melons, lettuces, tomatoes, leeks, green onions, rice, roses and grasses.

Preferably, the antimicrobial microbial agent further comprises a microbial delivery medium for the antimicrobial microbial strain.

Preferably, the microbial delivery medium comprises 40 to 65% by weight of bran, 1 to 5% by weight of chitosan, 30 to 55% by weight of sawdust, 1 to 3% by weight of chitin and 1-3% by weight of protein medium derived from cotton seed. do. More preferably, the microbial delivery medium further comprises 0.2 to 3.5% by weight spore production medium relative to the total weight of the microbial delivery medium. The medium is not particularly limited as long as it can produce spores, but it is preferable to use ATCC # 5 medium or YGM medium.

The antimicrobial microbial strain is preferably 1.0x10 ⁵ to 10 ¹⁰ , preferably 10 ⁷ to 10 ⁸ as a colony forming unit (cfu) per 1g of the microbial delivery medium.

Preferably, the microbial delivery medium comprises 1.0 to 3.0% by weight of pectin, 0.1 to 0.6% by weight of colloidal chitin and the remaining amount of water, and is in liquid form. Preferably, the antimicrobial microbial strain contains 10 ⁵ to 10 ¹⁰ , preferably 10 ⁷ to 10 ⁸ cfu per 1 ml of the liquid microbial delivery medium.

More preferably the microbial delivery medium comprises 0.2% by weight of pectin and is in liquid form. Preferably, the antimicrobial microbial strain contains 10 ⁴ to 10 ⁸ cfu per 1 ml of the liquid microbial delivery medium.

In order to achieve the above another technical problem, the present invention comprises the steps of culturing at least one antimicrobial microbial strain of Streptomyces genus WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) Provided is a method for preparing an antimicrobial microbial agent.

Preferably, the incubation is under shaking for 3 to 7 days at 25 to 33 ° C.

After obtaining the cells, the cells are lyophilized using a freeze dryer to prepare a cell powder or further mixing with an appropriate microbial delivery medium.

According to another aspect of the present invention, another technical task of the present invention is a protein medium derived from 40 to 65% by weight of bran, 1 to 5% by weight of chitosan, 30 to 55% by weight of sawdust, 1 to 3% by weight of chitin and cottonseed. Uniformly mixing 1 to 3% by weight and sterilizing to form a microbial delivery medium, at least one of Streptomyces genus WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) Mixing the antimicrobial microbial strain culture and incubating at 25 to 33 ° C. for 5 to 14 days and aseptically drying the resultant in a laminar flow bench sterilized with ultraviolet rays at room temperature. It is achieved by a method for producing an antimicrobial microbial agent comprising.

Preferably, after the drying step, the resultant further comprises the step of aseptically blending and grinding to 60 mesh in a laminate flow bench sterilized with ultraviolet rays using a blender surface sterilized with Freon gas.

Preferably, after the step of forming the microbial delivery medium, the microbial delivery medium is molded into pellets using a conventional extruder, and then 0.2 to 3.5% by weight of the spore generating medium with respect to the total weight of the obtained pellets. Further comprising coating in an amount.

In the mixing of the antimicrobial microbial strain, the antimicrobial microbial strain is mixed with 1.0 × 10 ⁵ to 10 ¹⁰ , more preferably 10 ⁷ to 10 ⁸ as cfu per gram of the microbial delivery medium.

Sterilization in the step of forming the microbial delivery medium is preferably heat sterilization for 30 to 40 minutes at 121 ℃.

According to another aspect of the present invention, another technical problem of the present invention is to uniformly mix 1.0 to 3.0% by weight of pectin, 0.1 to 0.6% by weight of colloid chitin and the remaining amount of water, and then sterilize to form a liquid microorganism delivery medium. And mixing at least one antimicrobial microbial strain culture medium of Streptomyces genus WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) to the microorganism delivery medium. It is achieved by the method of preparation of the formulation.

Preferably, the antimicrobial strain is mixed with 1.0 × 10 ⁵ to 10 ¹⁰ , preferably 10 ⁷ to 10 ⁸ cfu per 1 ml of the liquid microbial delivery medium.

In another aspect, the present invention provides a method of treating a microbial agent, wherein the microbial agent is coated, mixed or sprayed on at least one of a seed, a potting compound, a plant, and a soil in which the plant is growing. It is achieved by a process for treating a microbial agent characterized in that.

The antimicrobial microbial agent of the present invention is an antimicrobial microbial strain, characterized in that it comprises at least one of the novel strains of Streptomyces genus WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0342BP), but similar to the strain of the present invention It is to be understood that microbial preparations obtained using the microbial delivery medium together with strains having antimicrobial properties also fall within the scope of the present invention.

Hereinafter, the present invention will be described in detail.

1.Types and Compositions of Mediums Used in the Present Invention

In the present invention, the following media were used, and they were all sterilized at 121 ^° C. for 15 minutes before use according to a conventional media sterilization method.

1) casamino acid / yeast extract / dextrose (CYD) agar medium 0.5g / l casamino acid (Difco), 0.8g / l yeast extract (Difco) per 1.0 liter of distilled water, 0.4g / l dextrose, K ₂ HPO ₄ 2.0 g / l and agar 18 g / l, pH was adjusted to 7.2-7.4.

2) WYE (water / yeast extract) agar medium was prepared with yeast extract 0.25g / l, K ₂ HPO ₄ 2.0g / l and agar 18g / l, pH was adjusted to 7.2 to 7.4.

3) Chitin agar medium contains 4-6 g of colloidal chitin (Hsu and Lockwood., Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil.Applied Microbiology. P. 422-426 , 1975), 6 g of (NH ₄ ) ₂ SO ₄ , 2 g of K ₂ HPO ₄ , 0.4 g of MgSO ₄ · 7H ₂ O, 0.3 g of NaCl, 0.1 g of CaCO _3, 1.0 ml of microsaline solution and 20 g Prepared with agar of, pH was adjusted to 7.0 to 7.2. Here, the trace salt solution was prepared with 0.1 g of FeSO ₄ · 7H ₂ O, 0.1 g of MnCl ₂ · 4H ₂ O and 0.1 g of ZnSO ₄ · 7H ₂ O in 100 ml of distilled water.

4) Laminarin (laminarin) agar medium was prepared with laminarin (0.25w / v%) in place of the colloidal chitin of chitin agar medium, the pH was adjusted to 7.2 to 7.4.

5) Modified bennett liquid medium was prepared with 2 g yeast extract, 2 g beef extract, 2 g peptone, 10 g glucose and 5 μg / ml in 1 liter of distilled water, pH 6.5-7.5 Adjusted to.

6) Modified Bennett agar medium was prepared in a modified Bennett liquid medium at 2.0w / v% agar.

7) ISP # 2 medium: A medium prepared by Difco was used.

8) ISP # 3 medium: A medium prepared by Difco was used.

9) ISP # 4 medium: A medium prepared by Difco was used.

10) Spore Formation (ATCC # 5; Bacterial and Bacterial Phage ATCC Catalog 17th Edition) Liquid Medium: 1.0 g yeast extract, 1.0 g beef extract, 2.0 g tryptose, 0.01 g FeSO ₄ , in 1 liter of distilled water Prepared with 10 g of glucose.

11) YGM (Yeast Extract / Glucose / Inorganic Salt) Medium: 6 g yeast extract, 10 g glucose, inorganic salt (5.3 g Na ₂ HPO ₄ , 1.98 g KH ₂ PO ₄ , 0.2 g in 1 liter of distilled water) MgSO ₄ · 7H ₂ O, 0.2g NaCl, 0.05g CaCl ₂ · 2H ₂ O) and 1.0 ml of microelements (Pridham Gottlieb, The utilization of carbon compounds by some actinomycetes as an aid for species determination.J Bacteriol. 56: 107-114. 1948). The trace element solution was prepared in 100 ml of distilled water with 0.64 g of CuSO ₄ · 5H ₂ O, 0.11 g of FeSO ₄ · 7H ₂ O, 0.79 g of MnCl ₂ · 4H ₂ O, 0.15 g of ZnSO ₄ · 7H ₂ O.

2. Identification of Streptomyces spp. WYE 20 and WYE 324

Streptomyces strains WYE 20 and WYE 324 are described in the Burgy's Mannual of Systematic Bacteriology (1989), the International Streptomyces Project (ISP) (1974), and William et al. (Williams, ST et al. , "A probability matrix for identification of Streptomyces", J. Gen. Micorbiol. 129: 1815-1830, 1983, "Numerical classification of Streptomyces and related genera", J. Gen. Microbiol. 129: 1743-1813, 1983). Morphological, physiological and chemical properties were investigated and identified. The culture and morphological characteristics were obtained by culturing using ISP medium and observing the mycelia and spores using an optical microscope and an electron microscope.

3. Preparation of strain culture solution for inoculation and storage of strains

A culture solution obtained by inoculating spores on a CYD agar medium in 500 ml Erlenmeyer flasks containing 50 ml of modified Bennett liquid medium was shaken at 25 ^o C to 33 ^o C, pH 6.5 to 7.5, 130 to 300 rpm for 1 to 4 days. It was used as strain culture medium for inoculation.

The strain was incubated at 25 ^o C until spores were formed using CYD agar for storage of the strain, and then stored at 4 ^o C and subcultured every 4 weeks. As a long-term storage method, a spore suspension was made in 15 to 30% glycerol sterilized at 121 ° C. for 15 minutes, and then stored at −70 ° C.

4. Production of Mycelia and Spore Cells by Shake Culture

Mycelia and spore cell production of Streptomyces sp. Strains WYE 20 and WYE 324 were inoculated with 8 ml of the strain culture in a 1.0 liter Erlenmeyer flask containing 200 ml of modified Bennett liquid medium or 200 ml of YGM medium, followed by 25 ^o C to 33 ^o C, pH 6.5-7.5, 130-300 rpm shaking by incubation for 3 to 7 days. If necessary, the culture solution was centrifuged at 4,000 rpm for 10 minutes, followed by aseptic manipulation to recover mycelia and spore cells.

5. Production of Mycelia and Spore Cells by Microbial Delivery Media and Preparation of Antimicrobial Microbial Agents

(1) Production of powdered microbial delivery medium, mycelia and spore cells and production of powdered microbial preparation using the same.

First, pharmamedia (Pharmamedia, The Budkeye Oilseed Products Company, Fort Worth) as protein medium derived from 40 to 65% by weight of bran, 1 to 5% by weight of chitosan, 30 to 55% by weight of sawdust, 1 to 3% by weight of chitin and cottonseed , Texas, USA) 1 to 3% by weight uniformly mixed. The powder composition thus obtained was pelletized using an injection molding machine, and then uniformly sprayed with 0.2 to 3.5 wt% of ATCC # 5 medium or YGM medium as spore generating medium, and then subjected to wet heat sterilization at 121 ° C. for 30 to 40 minutes.

At this time, when the spore producing medium is added in less than 0.2% by weight, the addition effect of the spore producing medium is hardly seen, and when added in excess of 3.5% by weight, the initial growth of the microorganism is inhibited due to the high concentration of the microbial medium.

100 to 200 ml of the antimicrobial microbial culture medium (10 ⁵ to 10 ⁷ cfu / ml) recovered by liquid culture in the above-described method, the sterilized microbial delivery medium was inoculated uniformly. Subsequently, after incubating for 5-14 days at 30 ° C. according to the degree of growth of microorganisms, harvested, dried in a sterilized chamber (laminar flow) sterilized with ultraviolet rays, powdered by using a crusher surface sterilized with alcohol, Powdered microbial preparations were prepared.

(2) Production of liquid microbial delivery media, mycelia and spore cells using the same, and preparation of liquid microbial preparations.

1.0 to 3.0% by weight of pectin, 0.1 to 0.6% by weight of colloidal chitin and the remaining amount of water is uniformly mixed and then sterilized to form a liquid microbial delivery medium, and then recovered by liquid culture in the above-described method The culture liquid was mixed to prepare a liquid microbial preparation.

6. Fungal Pathogens

Fungal pathogens for antimicrobial testing include Pythium ultimum , Pythium aphanidermatum , Pythium graminicola , Rhizoctonia solani , and Ryezo . Joktoria Solani AG 1 (IB), Laytononia Solani AG 2-2 (IV), Fusarium oxysporum , Fusarium solani , Phytophthora Capsaish capsici ), Phytophthora parasitica , Sclerotinia sclerotiorum , Sclerotinia sclerotiorum , Verticillium dahliae , Rhizoctonia solani Kuhn (Rhizoctonia solani Kuhn), blood Aura now kyulre Ria (Pyicularia oryzae), Rhizoctonia seed trellis (Rhizoctonia cerealis), Loti's Claw Lake California Meo Kappa (Sclerotinia homoeocarpa), Colle Tote The Com gras mini-Cola (Colletotrichum graminicola), Colle Sat Com Nico tree Tia four (Colletotrichumnicotianae), Botrytis cine Ria (Botrytis cinerea) was used.

These fungal strains were incubated in PDA (Potato Dextrose Agar, Difco) medium or CMA (Corn Meal Agar, Difco) medium at 25 ^° C. and then stored at 4 ° C.

7. Determination of Cell Number of Microbial Agents

1.0 g of a powdered microbial agent was added to 99 ml of sterilized distilled water, mixed for 10 minutes to form a homogeneous suspension, and the number of cells was determined as colony forming units per g on CMA medium by continuous dilution and smearing. The cell number of the liquid microbial agent was also determined in the same manner.

8. Activity analysis of antimicrobial agents

In order to investigate the plant growth promoting activity and fungal pathogen control activity of the antimicrobial microorganisms, the antimicrobial microorganisms of the present invention were treated with cucumber and red pepper seeds, sown in soil or appropriate pots, and then germination rate, growth and growth of germinated plants, and The incidence rate reduction was measured, and the ability to control the onset of grass was also evaluated in comparison with the untreated control group.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited by the following examples.

Example 1: Isolation of Antimicrobial Microbial Strains

In order to isolate the microbial strains having excellent antimicrobial properties for use in the preparation of the antimicrobial microbial agent of the present invention, root samples of black pepper plants were collected from four locations located in Goesan-gun, Chungcheongbuk-do. The collected soil samples were dried at room temperature, suspended in 200 ml of sterile distilled water per 50 g of dried soil, diluted in sterilized distilled water to 10 ^-3 to 10 ^-6 and spread on WYE agar medium and incubated at 25 ° C. The resulting single colonies were separated in a UV lamina flow bench.

Pure separation of the isolated colonies was incubated for 4 to 10 days to form spores at 25 ^o C in passage to WYE agar medium was separated pure water by repeating the subculture on a new WYE agar medium.

Pure cultures were inoculated in CYD slope medium and then cultured at 25 ° C. to 30 ° C. to form spores, stored at 4 ° C., and passaged every 4 weeks.

The purely separated single colony was determined from the serial number of the WYE 1 by the method of determining the WYE serial number according to the serial number which is the sequence of WYE and the purely separated colony. That is, WYE 20 and WYE 324 are strains isolated from the 20th and 324th in the WYE agar medium through the pure separation process described above.

The strains obtained above were tested for antimicrobial activity, lysogenic cell wall degrading enzyme production, root engraftment growth, and low temperature growth (4 ° C., 8 ° C.).

Example 2: Identification and Characterization of Isolated Antimicrobial Strains

In order to identify the antimicrobial microorganism strains obtained in Example 1, the analysis of morphological, physiological and chemical properties was carried out according to the method of the buggy's manual. These results are shown in Tables 1-5.

Badge Growth High altitude mycelia Substrate hyphae Spore color WYE 20 WYE 324 WYE 20 WYE 324 WYE 20 WYE 324 WYE 20 WYE 324 ISP # 2 Good Good Hearty Hearty Light yellow Light yellow Gray pink Gray pink ISP # 3 Good Good Hearty Hearty Light yellow Light yellow Gray pink Gray pink ISP # 4 Good Good Hearty Hearty Light yellow Light yellow Gray pink Gray pink

Characteristics Strain WYE 20 Strain WYE 324 Spore characteristic: Spore chain form Spore surface form Spore colony color Spore shape Spirales Smooth gray pink (ISP # 2, # 3, # 4) Cylinder type Spirales Smooth gray pink (ISP # 2, # 3, # 4) Cylinder type Diffuse Pigment Production - - Melanin production + (Brown) + (Brown) Antibacterial activity: Aspergillus niger Bacillus cactils NCIB 3610 Streptomyces murine scandinavian albicans saccharomyces cerevisiae CBS 1172 micrococcus Luteus NCIB 196 +++-+ +++-+ Lecithinase activity + + Factine resolution - - Scheme Milk Hydrolysis + + Starch hydrolysis + + Nitrate reducing power + + H ₂ S production + ^{4 weeks} - Antibiotic Resistance (μg / ml): Oleandomycin (100) Neomycin (50) Rifampicin (50) Lincomycin (100) Novobiocin (100) Kanamycin (100) Ampicillin (100) Streptomycin (100) Cargogo Mycin (100) Tetracycline (100) Chloramphenicol (100) ^{-1mm low resolution} + ^{-2mm low resolution} + ^{-2mm low resolution} + ^{-3mm low resolution} ++-Tin:-: 4 mm or more inhibited ^{-1mm low resolution} +-+- ^{3mm low resolution} + ^{-2mm low resolution} - ^{1mm low resolution} + ^{-3mm low resolution} Tin: +: No growth inhibition zone

Characteristics Strain WYE 20 Strain WYE 324 Degradation activity: xanthine elastin arbutin xylan (xylan) L-tyrosine allantoin (allantoin) -+-+++ -+-+++ 45 ^o C growth - - Growth at pH 4.3 - -

Characteristics Strain WYE 20 Strain WYE 324 Growth in presence of growth inhibitory substance, (%, w / v): NaCl (7.0) Sodium azide (0.01) Phenol (0.1) Tellurium potassium (0.001) Crystal violet (0.0001) Neoline (0.5) Neoline (1.0) Twin 20 (0.5) Twin 20 (1.0) Relax (0.1) Relax (1.0) Chill Pepper (0.1) Smirex (0.1) Ridillo Mill (0.125) Ridillo Mill (0.25) Ancol (0.1) -++++++++++-+- -++-+++++++-±- Nitrogen source use (0.1% w / v): Asparagine DL-α-amino-n-butyric acid L-cysteine L-valine L-phenylalanine L-histidine L-hydroxyproline L-arginine L-methionine potassium nitrate L-serine L -Threonine +-±±-+++ ±-++ +-++ ± ++++-++

Characteristics Strain WYE 20 Strain WYE 324 Use of carbon source (1.0% w / v): Sucrose meso-inositol mannitol L-rhamnos raffinose D-melezitos adonitol D-melibiodextran xylitol L-arabinose D-fructose D- Galactose D-glucose D-salicylic D-xylosesobos (sorbose) D-lactose D-mannose halos maltose cellobioinulin sodium acetate (0.1) sodium citrate (0.1) sodium malonate (0.1) sodium propionate Sodium Pyruvate (0.1) +-+-+-±±±± +++ ± +++++++-++ ± ++ + ---- +-±-± +++++++++++-++ ± ++ Diaminopimelic acid LL LL

Fatty acid distribution Streptomyces spp. WYE 20 content (%) 1 time Episode 2 3rd time 4 times Minimum value Value medium Saturated fatty acid 14: 0 0.33 0.30 0.53 0.53 0.30 0.53 0.42 15: 0 1.52 1.54 1.13 1.19 1.13 1.54 1.35 16: 0 5.09 5.22 5.95 6.03 5.09 6.03 5.57 17: 0 0.30 0.31 - - nd nd nd Unsaturated fatty acid 16: 1 cis 9 6.42 6.25 5.45 5.80 5.45 6.42 5.98 17: 1 cis 9 1.08 1.06 0.65 0.63 0.63 1.08 0.86 The presence of a branch 13: 0 anteiso 0.14 0.14 - - nd nd nd 14: 0 iso 1.30 1.23 2.40 2.26 1.23 2.40 1.80 15: 0 iso 7.19 7.09 7.58 7.21 7.09 7.58 7.27 15: 0 anteiso 27.95 27.48 30.94 32.13 27.48 32.13 29.63 16: 1 iso H 2.80 2.69 3.01 2.98 2.69 3.01 2.87 16: 0 iso 15.01 15.17 17.71 17.09 15.01 17.71 16.25 16: 0 9? CH ₃ 3.42 3.42 2.77 2.69 2.69 3.42 3.08 17: 1 anteiso C 7.24 7.25 5.56 5.82 5.56 7.25 6.47 17: 0 iso 1.83 1.93 1.85 1.65 1.65 1.93 1.82 17: 0 anteiso 11.78 12.35 10.77 10.63 10.63 12.35 11.38 17: 0 10 CH ₃ 0.23 0.27 - - nd nd nd 18: 1 iso H 0.99 1.02 0.62 0.59 0.59 1.02 0.81 Presence of hydroxyl groups 17: 0 iso 2OH 0.40 0.41 - - nd nd nd 17: 0 3OH 0.27 0.32 - - nd nd nd Cyclopropane 17: 0 Cyclopropane 3.30 3.27 2.30 2.23 2.23 3.30 2.78 Unknown 17.595 SM 0.78 0.82 0.50 0.55 0.50 0.82 0.66

Note: Once and twice: Cell harvested from trypticase soy broth (TSB).

3 times, 4 times: Cell culture in tryticase soy agar (TSA).

-: Not detected. nd: not determined.

Fatty acid distribution Streptomyces spp. WYE 324 content (%) 1 time Episode 2 3rd time 4 times Minimum value Value medium Saturated fatty acid 14: 0 0.28 0.26 0.42 0.45 0.26 0.45 0.35 15: 0 2.22 2.19 1.23 1.25 1.23 2.22 1.72 16: 0 4.34 4.40 5.89 6.19 4.34 6.19 5.21 17: 0 0.40 0.43 - - nd nd nd Unsaturated fatty acid 15: 1 B 0.20 0.15 - - nd nd nd 16: 1 cis 9 6.40 6.39 5.66 5.54 5.54 6.40 6.00 17: 1 cis 9 1.40 1.43 0.78 0.71 0.71 1.43 1.08 A wooden branch 13: 0 iso 0.08 0.10 - - nd nd nd 13: 0 anteiso 0.13 0.14 - - nd nd nd 14: 0 iso 1.64 1.62 2.18 2.30 1.62 2.30 1.94 15: 0 iso 7.48 7.48 8.15 8.10 7.48 8.15 7.80 15: 0 anteiso 30.25 30.10 31.65 31.76 30.10 31.76 30.94 16: 1 iso H 2.59 2.57 3.34 3.23 2.57 3.34 2.93 16: 0 iso 14.05 14.06 16.67 16.69 14.05 16.69 15.37 16: 0 9 CH ₃ 3.56 3.56 3.29 3.12 3.12 3.56 3.38 17: 1 anteiso C 6.68 6.65 5.97 5.90 5.90 6.68 6.30 17: 0 iso 1.77 1.82 2.14 2.16 1.77 2.16 1.97 17: 0 anteiso 10.78 10.95 10.68 10.72 10.68 10.95 10.78 17: 0 10 CH ₃ 0.18 0.18 - - nd nd nd 18: 1 iso H 0.49 0.50 - - nd nd nd Presence of hydroxyl groups 17: 0 iso 2OH 0.50 0.51 - - nd nd nd 17: 0 3OH 0.24 0.19 - - nd nd nd Cyclopropane 17: 0 Cyclopropane 3.15 3.14 1.95 1.89 1.89 3.15 2.53 Unknown 17.595 SM 0.68 0.69 - - nd nd nd

Note: Once and twice: Cell harvested from TSB.

3, 4 times: Cell culture in TSA.

-: Not detected. nd: not determined.

division Streptomyces WYE 20 Streptomyces colombiensis ATCC 27425 Streptomyces WYE 324 Streptomyces goshikiensis ATCC 23914 Spore Chain: Rectiflexibles - + - - Spore Chain: Retinaculiaperti - - - - Spore Chain: Spiral / spirales + + + + Spore Chain: Verticillati - - - - Spore surface: smooth + + + + Spore surface: rugose - - - - Spore Color: Red (red) - + - + Spore Color: Gray (Gray) - - - - Spore Color: Green - - - - Diffuse Pigment: Red / Orange - - - - Diffuse Pigment: Yellow / Brown - - - - Melanin production + + + + Mycelial fragmentation - - - - DL-α-amino-n-butyric acid (0.1%, w / v) - - - - Whether to use L-histidine (0.1%, w / v) + - + - Whether to use L-hydroxyproline (0.1%, w / v) + + + - Lecithinase Activity + + + + Pectin hydrolysis - - - - Nitrate Reducing Power + + + + H ₂ S production + + - - Bacillus Certilis NCIB Antibacterial Activity + + + + Streptomyces murines antibacterial activity + + + + Aspergillus Niger Antibacterial Activity + + + + Xanthin resolution - + - + Allantoin resolution + + + + Arbutin resolution - + - - Neomycin (50 μg / ml) resistance + + + + Rifampicin (50 μg / ml) resistance - - - - 45 ℃ growth - - - - Growth in the presence of NaCl (7%, w / v) - - - - Growth in presence of azide sodium (0.01%, w / v) - - - - Growth in the presence of phenol (0.1%, w / v) + + + + Availability of D-xylose (1.0%, w / v) + - + - Availability of meso-inositol (1.0%, w / v) - - - - Availability of mannitol (1.0%, w / v) - - - - Use of D-fructose (1.0%, w / v) + + + + Whether to use L-Rhamnose (1.0%, w / v) + - - - Rafinose (1.0%, w / v) availability - - - - Inulin (1.0%, w / v) availability - - - - Use of Adonitol (1.0%, w / v) - - - - Cellobios (1.0%, w / v) availability + + + +

As a result of analyzing the results shown in Tables 1 to 5 according to the aforementioned Burgess manual and the method of ISP, William, etc., it was identified that two strains obtained in the present invention belong to Streptomyces genus. In other words, WYE 20 was identified as a new strain close to Streptomyces colombiensis belonging to Cluster 61 by analyzing the morphological, physiological and chemical properties. In addition, WYE 324 was found to be a new strain close to Streptomyces goshikiensis belonging to cluster 61. On the other hand, these strains WYE 20 and WYE 324 on January 9, 1997, the Gene Bank (Korean Collection for Type Cultures, in the Biotechnology Research Institute located at 52, Won-dong, Yuseong-gu, Daejeon, Republic of Korea, 305-333, according to the Treaty of Budapest). Accession numbers KCTC 0341BP (WYE 20) and KCTC 0342BP (WYE 324) were deposited with Korea Research Institute of Bioscience and Biotechnology.

Example 3: Optimum Growth Temperature Range Measurement Experiment

In order to determine the growth at the specific temperature and the optimum growth temperature range, after inoculating WYE 20 and WYE 324 of Streptomyces genus identified in Example 2 in modified Bennett agar medium, 4 ^o C, 8 ^o C, 27 ^o C The growth was observed every week while incubating at 37 ^o C and 45 ^o C for 3 weeks. The result is good growth (+) depending on the growth rate; Slow growth (±); It is shown in Table 6 evaluated by the lack of growth (-) and the like.

Strain 4 ℃ 8 ℃ 27 ℃ 37 ℃ 45 ℃ 7 days 21st 7 days 14 days 7 days 14 days 7 days 14 days 7 days 14 days WYE 20 ± + + + + + ± + - - WYE 324 - ± ± + + + ± + - -

As shown in Table 6 above, WYE 20 started to grow slowly after 1 week at 4 ^o C and grew well after 3 weeks, whereas strain WYE 324 did not start to grow slowly after 3 weeks at 4 ^o C. Strain WYE 20 is 8 ^o was well grown in both the C and 27 ^o C, when the strain WYE 324, 8 ^o C in the beginning to grow slowly after 1 week showed good growth after two weeks has elapsed, preferably the 27 ^o C I grew up. Both strains started to grow slowly after one week of incubation at 37 ^o C and grew well after two weeks of incubation, but did not grow at 45 ^o C.

Example 4: Activity and Antagonistic Assay of Enzymes

(1) Determination of enzyme activity

Chitinase and β-1,3-glucanase are known enzymes that play an important role in attacking and killing fungal pathogen mycelia. Therefore, these enzyme activities were measured for the novel strains of the present invention as follows.

Chitinase activity was confirmed by inoculating strains in chitin agar medium containing colloidal chitin as the only carbon source and culturing for 14 days at 30 ^o C to observe colony formation and its formation rate. As a result, both WYE 20 and WYE 324 of the present invention showed chitinase activity.

In order to measure β-1,3-glucanase activity, strains were inoculated into laminarine agar medium containing only laminarin as a carbon source, followed by incubation at 27 ^o C for 7 days to observe colony formation and its formation rate. It was. As a result, it was shown that both WYE 20 and WYE 324 of the present invention have very high β-1,3-glucanase activity.

(2) control pair antagonistic analysis

Antagonistic properties of pure strains WYE 20 and WYE 324 were determined by determining the extent of growth inhibition of fungal pathogens through plate control pair experiments.

Strains of the invention were inoculated in CMA medium and incubated at 30 ^° C. for 6-10 days. In the center of the plate was inoculated with PDA (Potato Dextrose Agar) block containing vigorously grown pathogen fungal hyphae and incubated at 25 ° C for 24 to 192 hours. On the other hand, for the control experiment, the same PDA block was inoculated on the CMA plate not inoculated with the strain of the present invention and cultured in the same manner. The results for the antagonistic experiment conducted as a growth inhibitory effect of the fungal pathogen by the strain of the present invention are shown in Table 7 below as the average value through three repeated experiments.

Host plants Fungal pathogens disease Control Pair Antagonism Measurement WYE 20 WYE 324 cucumber PC Ultimum ¹ Seedling disease, seed rot disease, root rot disease + + Ventgrass PC APANY THE METHOM ² PC Umbrite + + Ventgrass PC Graminicola ² PC Umbrite ++ ++ tomato Fusarium Oxy Room Rot Rot Disease, Wither Disease ++ ++ cucumber Fusarium Solani Mozalok disease ++ ++ cucumber Raiatonia Solani Mozalok disease ++ ++ Ventgrass Raiztonia Solani AG 1 (IB) Brown patches ++ ++ Georgia Japonica Raiztonia Solani AG 2-2 (IV) Large patch ++ ++ pepper Pitotovsora Capsaish Pepper Plague ++ ++ tomato Phytovsora Parasitikka Root rot ++ ++ Beans and lettuce Sclerotinia sclerotium ^a drop ++ ++ onion Sterlotium Sepibo Room ³ Black rot bacteria ++ ++ Cotton, alfalfa Verticilium Dahlia ³ Wilted ++ nt rice plant Raiatonia Solani Kuhn Paperback disease ++ ++ rice plant Picureria Orizee Blast ++ ++ Ventgrass Raiztonia Cirelis Yellow Patch (Winter Patch) ++ ++ Georgia Japonica Raiztonia Cirelis Spring Dead Spot ++ ++ Ventgrass Sclloretinia Homeocapa Dallas Spot ++ ++ Ventgrass Colletotricum Gramini Cola anthrax ++ ++ tobacco Colletotricom Nicotiane anthrax ++ ++ Strawberry, pumpkin, cucumber, tomato, pepper, green onion, leek, etc. Botrytis Cinema Gray Mold Disease ++ ++

^1: 24 hours, ^{236 hours, 3:} 192 hours, and the rest are all 96 hours of the culture observation

++: indicates an antagonistic inhibition zone of 1.0 cm or more

+: Shows obvious growth inhibition area near colony

-: Very weak or not antagonistic

n.t .: not tested

a: causes disease in about 408 plant species

As shown in Table 7, the strains WYE 20 and WYE 324 showed a clear growth inhibition zone near the colonies for the P. C. ultimateim, P. apanimederumtum, the strain WYE 20 is a C. Graminicola, Fusa Lysium oxysporum, Fusarium solani, lysatonia solani, lysatonia solani AG 1 (IB), lysatonia solani 2-2 (iv), phytopsora capsaish, phytopsora parasi Tikka, Sclerotinia sclerotiorum, Sclerotidium cepiborum, Bertisilum Dahlia, Raiztonia Solani Kuhn, Picureria Orizee, Raiztonia cerellis, Scleroti For Nia Homeocapa, Colletotricum Graminicola, Colletotricom Nicothiane and Botrytis Ceria, strain WYE 324 is composed of Pseum graminicola, Fusarium oxysporum, Fusarium solani, Raiatonia Solani, Raiatonia Solani AG 1 (IB), Raiztonia Solani 2-2 (IV), Phytopsora Capsaicy, Phytopsora Parasitika, Sclerotinia Sclerotium, Sclerotidium Sepivorum, Rai About Jotonia Solani Kuhn, Piculeria Orizee, Laytontonia Cerellis, Sclloretinia Homeocapa, Colletotricum Graminicola, Colletotricom Nicotiane and Botrytis Ceria Strong antagonism was shown.

Example 5 Preparation of Powdered Microbial Preparation

In order to produce a powdered microbial agent suitable for maintaining the viability of the microbial strain of the present invention and to enable the treatment of seeds, roots or soil of plants, an appropriate powdered microbial delivery medium was first prepared.

To this end, the mixture was changed while changing the mixing ratio of bran, chitosan, sawdust, chitin, and Pharmamedia to form a powder composition, which was then molded into pellets. The pellets were molded by uniformly mixing in the proportions shown in Table 8 below. In this process, the forming of the pellets is investigated, and when forming into pellets without shattering in the forming process, it is "good", or when shattering occurs and it is disturbed to be formed into pellets, as a result, It is shown in Table 8 below. In addition, the degree of air permeability and cell growth of the microbial delivery medium prepared in each case was also investigated, and the relative results were divided into three grades, and the results were shown as "low", "normal", " Good ".

Microbial delivery medium prepared as described above was uniformly inoculated antimicrobial microbial strain samples obtained by liquid culture in the above-described method (10 ⁵ to 10 ⁷ cfu / ml). This was incubated at 30 ° C. for 5 to 14 days according to the growth of microorganisms, and then dried in an ultraviolet sterilized lamina flow and powdered using a pulverizer ground sterilized with alcohol to prepare a high concentration powder type microbial preparation. . At this time, the antimicrobial microbial strain was adjusted to exist 1.0 × 10 ⁵ to 10 ¹⁰ as cfu for 1g of the powdered microbial delivery medium. The samples thus obtained were divided into two groups for each microbial strain and stored for 3 months at 4 ° C and 25 ° C. During storage, the viability of microbial strains and the regeneration activity of viable microorganisms were measured at 1 month intervals. Specifically, 1 g of each of the finally obtained samples was taken, dissolved in 9 ml of sterilized distilled water, and the supernatant was measured for colony forming units (cfu) and the regrowth of viable cells was measured. Table 9 shows.

For comparison, a microbial delivery medium was prepared in the same manner as described above, except that the composition for each component of the microbial delivery medium was mixed at a ratio as described in Table 8 below. The characteristics of the branches were evaluated in the same manner as described above, and the results are shown in Table 9 below.

(Unit: weight%) division bran Chitosan sawdust Chitin Pharmamedia Pellet Molding Aeration degree Cell growth Experiment group 1 65 One 30 2 2 Good usually usually Experiment group 2 65 2 30 One 2 Good usually usually Experiment group 3 65 2 30 2 One Good usually usually Experimental Group 4 65 One 30 One 3 Good usually usually Experimental group 5 65 One 30 3 One Good usually usually Experimental Group 6 63 5 30 One One Good usually usually Experimental group 7 60 5 30 2 3 Good usually usually Experimental Group 8 50 One 45 2 2 Good Good Good Experimental Group 9 45 5 45 2 3 Good Good Good Experimental group 10 45 3 50 One One Good Good Good Experimental Group 11 40 3 55 One One Good Good Good Experimental Group 12 40 One 55 3 One Good Good Good Experimental Group 13 40 One 55 One 3 Good Good Good Control group 1 90 2 5 2 One Good Low Low Control 2 80 5 10 3 2 Good Low Low Control group 3 70 3 25 One One Good Low Low Control 4 30 5 60 2 3 Low Good nt Control group 5 20 5 70 2 3 Low Good nt Control group 6 10 5 80 2 3 Low Good nt

nt: not tested

(Unit: weight%) division bran Chitosan sawdust Chitin Pharmamedia Initial cell count (cfu / g) of WYE 20 or WYE 324 Cell count of WYE 20 or WYE 324 after 3 months (cfu / g) Regeneration Field of Surviving Cells 4 ℃ storage 25 ℃ storage Experiment group 1 65 One 30 2 2 1.2 x 10 ⁷ 1.0 x 10 ⁶ 2.3 x 10 ⁵ Good Experiment group 2 65 2 30 One 2 1.9 x 10 ⁷ 1.5 x 10 ⁶ 2.9 x 10 ⁵ Good Experiment group 3 65 2 30 2 One 1.3 x 10 ⁷ 1.4 x 10 ⁶ 2.5 x 10 ⁵ Good Experimental Group 4 65 One 30 One 3 2.1 x 10 ⁷ 1.7 x 10 ⁶ 3.6 x 10 ⁵ Good Experimental group 5 65 One 30 3 One 2.0 x 10 ⁷ 1.2 x 10 ⁶ 3.1 x 10 ⁵ Good Experimental Group 6 63 5 30 One One 2.7 x 10 ⁷ 1.1 x 10 ⁶ 2.7 x 10 ⁵ Good Experimental group 7 60 5 30 2 3 1.5 x 10 ⁷ 1.3 x 10 ⁶ 3.3 x 10 ⁵ Good Experimental Group 8 50 One 45 2 2 1.6 x ^{10 8} 2.1 x 10 ⁷ 2.1 x 10 ⁶ Good Experimental Group 9 45 5 45 2 3 2.6 x ^{10 8} 1.1 x 10 ⁷ 2.5 x 10 ⁶ Good Experimental group 10 45 3 50 One One 2.1 x ^{10 8} 1.0 x 10 ⁷ 3.1 x 10 ⁶ Good Experimental Group 11 40 3 55 One One 1.9 x ^{10 8} 1.6 x 10 ⁷ 3.5 x 10 ⁶ Good Experimental Group 12 40 One 55 3 One 1.8 x ^{10 8} 1.9 x 10 ⁷ 2.4 x 10 ⁶ Good Experimental Group 13 40 One 55 One 3 2.2 x ^{10 8} 1.3 x 10 ⁷ 1.9 x 10 ⁶ Good

From Tables 8 and 9, when the components of the microbial delivery medium are mixed within the range defined in the present invention (Experimental Group 1 to 13), the moldability to the pellets is good, and the air permeability and cell growth are more than normal. It can be maintained as, but when mixed in a range outside the scope defined in the present invention (controls 1 to 6), any one of the moldability, breathability or cell growth into pellets was found to be particularly poor.

In addition, in order to determine the coating effect of the spore generating medium in the preparation of the microbial delivery medium, spray coating the YGM medium as the spores production medium in the ratio shown in Table 10 for each microbial delivery medium of the experimental groups 1 to 13 and then 121 ℃ Wet heat sterilization at 30 to 40 minutes. After preparing a microbial preparation using the microbial delivery medium prepared in this way, the characteristics thereof are shown in Table 10 below.

(Unit: weight%) division bran Chitosan sawdust Chitin Pharmamedia YGM ^* Initial cell count (cfu / g) of WYE20 or WYE 324 Cell count of WYE 20 or WYE 324 after 3 months (cfu / g) Regeneration Field of Surviving Cells 4 ℃ storage 25 ℃ storage Experimental group 14 65 One 30 2 2 0.2 1.1 x 10 ⁷ 1.2 x 10 ⁶ 2.4 x 10 ⁵ Good Experimental group 15 65 2 30 One 2 0.5 1.4 x 10 ⁷ 1.4 x 10 ⁶ 2.9 x 10 ⁵ Good Experiment Group 16 65 2 30 2 One 1.0 1.6 x 10 ⁷ 1.6 x 10 ⁶ 2.6 x 10 ⁵ Good Experimental Group 17 65 One 30 One 3 1.5 2.0 x 10 ⁷ 1.9 x 10 ⁶ 4.2 x 10 ⁵ Good Experimental Group 18 65 One 30 3 One 1.8 2.2 x 10 ⁷ 1.3 x 10 ⁶ 3.2 x 10 ⁵ Good Experimental Group 19 63 5 30 One One 2.0 1.7 x 10 ⁷ 2.1 x 10 ⁶ 3.7 x 10 ⁵ Good Experimental Group 20 60 5 30 2 3 3.5 1.1 x 10 ⁷ 2.3 x 10 ⁶ 3.4 x 10 ⁵ Good Experimental group 21 50 One 45 2 2 3.5 1.2 x ^{10 8} 2.3 x 10 ⁷ 2.3 x 10 ⁶ Good Experimental Group 22 45 5 45 2 3 2.5 1.6 x ^{10 8} 2.1 x 10 ⁷ 2.5 x 10 ⁶ Good Experimental Group 23 45 3 50 One One 2.0 2.2 x ^{10 8} 1.3 x 10 ⁷ 3.2 x 10 ⁶ Good Experimental Group 24 40 3 55 One One 1.5 2.9 x ^{10 8} 2.1 x 10 ⁷ 3.6 x 10 ⁶ Good Experimental Group 25 40 One 55 3 One 0.5 1.4 x ^{10 8} 1.9 x 10 ⁷ 2.6 x 10 ⁶ Good Experimental Group 26 40 One 55 One 3 0.2 1.2 x ^{10 8} 1.3 x 10 ⁷ 2.9 x 10 ⁶ Good

YGM ^* :% by weight of the sum of all remaining

Table 9 and Table 10, it can be seen that the microbial agent prepared using the microbial delivery medium of the experimental group is excellent in the characteristics that can maintain the cells in a stable state for a long time. That is, even after three months, the microbial strain cell number of all the samples of the experimental group was maintained at 10 ⁵ cfu / g or more necessary to express the titer as a microorganism agent, in particular the microbial delivery medium prepared by coating the spore producing medium of the experimental group 14 to 26 In the case of using this activity was shown to be more enhanced.

The powdered microbial preparation prepared in this way was excellent in storage at room temperature and was observed to maintain cell numbers of 10 ⁵ cfu / g or more, which is the number of microbial strains required to maintain minimal microbial activity even after 3 months at room temperature.

Example 6: Preparation of Liquid-Type Microbial Formulation (I)

20 g of pectin, 2 g of colloidal chitin, and the remaining amount of water were uniformly mixed to form a 1 liter suspension, which was then sterilized for 15 minutes at 121 ° C. in a conventional manner. Subsequently, the microorganisms and spores of the antimicrobial microbial strain Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) obtained by liquid culture in a sterile liquid microbial delivery medium were prepared by the above-described method. (1.2 × 10 ⁷ cfu / ml) Liquid microbial formulations were prepared.

The liquid microbial preparation thus obtained was stored at 4 ° C. Liquid microbial preparations are suitable for the irrigation of seedlings seedlings or seedlings, and also effective for diluting with water and spraying.

Example 7: Preparation of Liquid Microbial Formulation (II)

Pectin (final pectin content 0.2 w / v%) sterilized in cell culture fluid of WYE 20 (KCTC 0341BP) of Streptomyces or WYE 324 (KCTC 0342BP) of Streptomyces obtained by liquid culture in the above manner By mixing (1.0 × 10 ⁶ -10 ⁸ cfu / ml) to prepare a liquid microorganism. Pectin was dissolved in water and sterilized by autoclaving at 121 ° C. for 15 minutes in a conventional manner.

The liquid microbial agent thus prepared was stored at 4 ° C. Liquid microbial preparations are suitable for the irrigation of seedlings seedlings and seedlings, diluted with water, and very effective for spraying.

Example 8: Activity of Antimicrobial Microbial Agents

The activity of the powdered microbial preparation of Example 5 (especially Experimental group 22 of Example 5) and the liquid microbial preparation (I) prepared according to Example 6 and stored at 4 ° C. was either directly treated with plants or seeds or roots or seedlings. It was measured as a method of promoting plant growth and controlling of fungal pathogens by indirectly mixing with the mother leaf, pot, potting compound or soil.

(1) Analysis of the Control Activity of Lysatonia Solani Mozalok Disease of WYE 20 (KCTC 0341BP) in Streptomyces by Cucumber Cultivation

Cucumber plants were measured for Lycitonia solani mozzarlock disease control activity of WYE 20 (KCTC 0341BP) through port seeding analysis.

Cucumber seeds were soaked in 20 ml (WYE 20 1.2 × 10 ⁷ cfu / ml) liquid microbial preparation (I) prepared in Example 6 for 3 hours and then seeded in pots. As an untreated control, seed was immersed in sterile water for 3 hours and liquid microorganism delivery medium for 3 hours.

As a potting formulation, Hortus (Hortus, UK) was sterilized at 121 ^o C for 60 minutes, cooled to room temperature, left for 12 hours and sterilized three times by sterilization at 121 ^o C for 60 minutes. Cucumber seeds were used for sowing.

As a pathogen, lysatonia solani which was cultured for 14 days (25 ^o C) in PDB (Potato dextrose broth: Difco) was recovered, and mixed with sterilized soil so that the incidence was about 60% in the untreated control group. After the soil was made, cucumber seeds of the treated experimental group and the control group prepared by the method described above were sown to measure the activity of each microbial agent (I).

As a seeding pot, a disposable paper cup having a diameter of 9 cm was used, three seeds were seeded per pot, and six cups were prepared for each treatment group. After seeding the pot was arranged in a glass greenhouse in a random block arrangement, the humidity was adjusted to maintain 40 to 60% and water was periodically supplied as needed. In addition, the temperature was maintained at 25 ^o C to 30 ^o C, the light used natural light.

Periodically, the germination of seeds and the onset of mozalococcal disease were observed and the final results are shown in Table 11.

Treatment Category Soybean Seeds Germinated cucumber seeds (%) Number of healthy plants (%) Body weight (g) / ground plant 7 days 14 days 18 days Pathogen free + no control 18 18b ^x (100) 18b ^x (100) 2.61b ^x Raiztonia Solani + Untreated Control 18 7a (39) 7a (39) 1.73a Raiztonia Solani + Liquid Microbial Delivery Control 18 8a (44) 8a (44) 1.75a Raiztonia solani + liquid microbial treatment experimental group 18 18b (100) 18b (100) 2.51b

^x Statistically comparing the results of the untreated control and treated experimental groups of the same column

If they differ in the range p = 0.05, they are represented by different alphabets (a, b).

As shown in Table 11, in the case of cucumber seeds treated with WYE 20 of the present invention, that is, the microbial agent (I) of the present invention, 100% of the onset of cucumber mozalok disease caused by Lysatonia solani was suppressed. In addition, in the untreated control group, growth disorders caused by lyxtonia solani were very severe, but in the experimental group treated with the microbial agent (I) of the present invention, there was no growth disorders by lyxtonia solani. . This indicates that the microbial agent (I) of the present invention is very effective in controlling the fungal pathogen Lyactonia solani and promoting plant growth.

(2) Cucumber powdery mildew control activity assay

Biologically Controlled Activity Analysis on the Inhibition of Occurrence of Powdery mildew in Cucumis sativus L. Crop in a Pot to Test the Efficacy of WYE 324 (1.2 x 10 ⁷ cfu / ml), the Microbial Agent (I) Prepared in Example 6 Was performed.

As a seeding pot, a disposable paper cup (9 cm in diameter) was used. One cucumber seed was sown in each cup containing a potting formulation consisting of agricultural soil and Hotus (UK) (4: 1 by volume). The cup was placed and maintained in a glass greenhouse.

20 mL of the microbial formulation containing WYE 324 was sprayed onto one cucumber crop (14 days old) at the beginning of the experiment and once more a week later. For crops in the control group water was sprayed in equal amounts. Three cucumber crops (one cucumber crop per cup) in each treatment and control group were prepared for analysis.

In order to spontaneously induce cucumber powdery mildew caused by Sphaerotheca fuliginea , three cucumber crop cups and three affected cucumber crops (one crop per cup) were used in each group, and random block arrays were installed in glass greenhouses. . Moisture was allowed to be maintained between 70% and 90% and water was supplied as needed. The temperature in the glass greenhouse was kept between 25 ° C and 30 ° C. The development of cucumber powdery mildew was investigated. This experiment was continued for two weeks, and the analysis was repeated. The results are shown in Table 12.

Treatment Cucumber powdery mildew Experiment 1 Experiment 2 Control + + WYE 324 treatment group - -

+: Powdery mildew. -: Powder powder does not occur. * There is a powdery mildew control effect which is statistically significant (p = 0.05) between control and treatment.

As shown in Table 12, powdery mildew was not found in cucumber crops sprayed with microbial preparations containing WYE 324. On the other hand, powdery mildew was observed in the control crops. These results showed that WYE 324 was effective in controlling powdery mildew in Cucumis sativus L.

(3) Analysis of Rhizoctonia Brown Patch Control Activity of Golf Course Green Grass

The activity of the microbial agent (I) of the present invention was measured by carrying out the control of the onset of Raiztonia brown patch of golf course green grass.

As the antimicrobial microorganism strain of the microbial agent (I) used in this Example was harvested and used spores and mycelia cell culture medium prepared by the above-mentioned shaking culture method, immediately before the treatment, Streptomyces strain WYE 20 of liquid microbial agent (I) Or the cell number of WYE 324 was sprayed adjusted to 2.0 x 10 ⁵ cfu / ml. As an experimental group and a control group, test blocks (1.5m × 1.5m) were set, and four test blocks (test blocks 1 and 2) were installed on two adjacent greens, respectively, for each treatment section. At this time, the test sphere was set in a random block array method. For experimental block, liquid microbial formulation (I) containing strain WYE 20 or WYE 324 was sprayed 2.5 liters per test zone at intervals of 7-10 days from June 25, 1996 to August 23, 1996, and the control group. The same amount of water was sprayed on the blocks. No chemical disinfectant was used during the test. The incidence of Layzatonia Brown patches on the greens was periodically observed and the incidence rates are shown in Table 13 (Test 1) and Table 14 (Test 2).

Treatment Raiztonia Brown Patch Incidence (%) 7/25/96 8/6/96 8/23/96 Untreated control group (WYE 20) Experiment group (WYE 324) 10.18a ^x 3.38b0.00c 14.6a ^x 9.2b5.2c 59a ^x 24b24b

^x Statistically comparing the results of the untreated control and treated experimental groups of the same column

If they differ in the range p = 0.05, they are represented by different alphabets (a, b, c).

Treatment Raiztonia Brown Patch Incidence (%) 7/25/96 8/6/96 8/19/96 Untreated control group (WYE 20) Experiment group (WYE 324) 5.6a ^x 0.0b0.0b 9.3a ^x 0.8b0.0b 28.3a ^x 5.3b0.7c

^x Statistically comparing the results of the untreated control and treated experimental groups of the same column

If they differ in the range p = 0.05, they are represented by different alphabets (a, b, c).

As shown in Table 13 (Test Tool 1) and Table 14 (Test Tool 2), each test tool 1 when treated with the microbial agent (I) of the present invention (including WYE 20 or WYE 324, respectively) And the incidence 2 significantly reduced the incidence of Raiztonia brown patches. This indicates that the microbial agent (I) of the present invention is very effective in controlling the onset of Lysatonia brown patches of green grass.

(4) Analysis of Rhizoctonia Large Patch Control Activity of Golf Course Fairway Grass ( Zoysia japonica )

To test the efficacy of the microbial preparation (I) comprising the Streptomyces genus WYE 20 or IBT 678 prepared in Example 6, a control activity assay for the inhibition of Lysatonia large patch of golf course fairway grass was performed. In microbial preparation (I), the cell number of WYE 20 or IBT 678 in Streptomyces was adjusted to 2.0 × 10 ⁶ cfu / ml.

Soil infected with Raiztonia solani AG 2-2 (IV) was inoculated into the fairway grass and transplanted into pots (25 cm in diameter). 200 ml of the microbial agent (I) of the present invention were treated for two weeks and three weeks at intervals of one week in 1996 and 1997, respectively. Pots treated with the same amount of water were used as controls. Three ports were used for each treatment and random block array was installed. To induce spontaneous outbreaks, the port was placed near the fairway of the golf course from early September to early October 1996 and from early March to late June 1997 to observe the development of Raiztonia large patches. As shown in Table 15, no large patch was observed in the pot treated with the microbial agent of the present invention including WYE 20, while a large patch disease was observed in the pot treated with the control and IBT 678.

Treatment Large patch onset of golf course fairway grass ( Zoysia japonica ) Fall 1996 1997 spring Pathogen + No Treatment + + Pathogen + WYE 20 Treatment - - Pathogen + IBT 678 Treatment Not tested +

+: Large patch disease outbreak-: Large patch disease does not occur.

As shown in Table 15, these results showed that WYE 20 was effective in controlling lyxtonia large patches of golf course fairway grass ( Zoysia japonica ).

(5) Analysis of Control of Red Pepper Diseases by Microbial Preparation through Pepper Cultivation

The pepper plant disease control activity of the microbial preparation (I) (including WYE 20 or WYE 324) of the present invention was measured by pot seeding analysis on pepper plants.

Pepper seeds, which were immersed in sterile water for 2 days, were immersed in liquid microbial preparation (I) of the present invention for 18 hours (experimental group). Immediately before seed treatment, the cell numbers of Streptomyces strains WYE 20 and WYE 324 of the microbial agent (I) were adjusted to 1.2 × 10 ⁷ cfu / ml and 1.7 × 10 ⁷ cfu / ml, respectively. As an untreated control, pepper seeds immersed in sterile water for 2 days were immersed in fresh sterile water for 18 hours. On the other hand, it was also compared with the immersion in the liquid microbial delivery medium prepared according to the method of Example 6 for 18 hours.

As in the experimental method through the cucumber cultivation, the recovery of the phytophorus capsaish cultured (25 ^o C) for 14 to 21 days in PDB (Potato dextrosebroth: Difco), the incidence of the untreated control is about 80% or more After mixing the sterilized soil to make a diseased soil, soybean seeds of the experimental group and the control group were sown to measure the pepper blight control activity of the microbial agent of the present invention.

As a seeding pot, a disposable paper cup having a diameter of 9 cm was used, three seeds were seeded per pot, and 14 seeding pots were prepared for each treatment group. The seeded pots were arranged in a glass greenhouse using a random block array method, the humidity was maintained at 80%, and water was periodically supplied as needed. In addition, the temperature was maintained from 25 ^o C to 32 ^o C, the light used natural light.

Periodically, the germination of seeds and the onset of pepper blight were observed and recorded in Table 16.

Treatment Category Soybean seed sown Germinated red pepper seeds (germination rate:%) Plague Seedlings (Incidence:%) 14 days 14 days 18 days Pathogen free + untreated control 42 37 (88a ^x ) 0 (0c ^x ) 0 (0c ^x ) Phytoprosora capsaish + untreated control 42 34 (81a) 28 (82a) 33 (97a) Phytoporacapsaicy + Liquid Microbial Media Delivery Control 42 35 (83a) 29 (83a) 34 (97a) Phytopora capsaisi + microbial agent (WYE 20) treatment experimental group 42 34 (81a) 12 (35b) 26 (76b) Pytopsora capsaishi + microbial agent (WYE 324) treatment experimental group 42 36 (86a) 22 (61c) 28 (78b)

^x Statistically comparing the results of the untreated control and treated experimental groups of the same column

If they differ in the range p = 0.05, they are represented by different alphabets (a, b, c).

As shown in Table 16, the pepper seeds treated with the microbial agent (I) (WYE 20 or WYE 324) of the present invention significantly inhibited the onset of pepper blight caused by phytophore capsaicy and showed statistical significance. It was. This indicates that the microbial agent (I) of the present invention is very effective in inhibiting the activity of the phytophthora capsaicin, which is a pepper pest germ.

(6) Analysis of growth promotion activity of microbial preparations through pepper cultivation

The pepper plant growth promoting activity of the microbial preparation of the present invention was measured through a pot seeding analysis on the pepper plants.

Pepper seeds soaked in sterile water for 2 days were treated with the powdered microorganism preparation prepared according to Example 5 (1000 seeds / 4 g). Immediately before seed treatment, the cell numbers of Streptomyces strains WYE 20 and WYE 324 of the microbial agent were adjusted to 1.2 x 10 ⁷ cfu / ml and 1.7 x 10 ⁷ cfu / ml, respectively. As an untreated control group, pepper seeds immersed in sterile water were used in the same manner. On the other hand, the same experiment was performed for the pepper seeds treated with the powdered delivery medium (1000 seeds / 4g) of the same composition not containing the antimicrobial microorganism strain of the present invention and compared the results.

Sowing of red pepper seeds was carried out in the same manner as in the cucumber cultivation experiment.

As a seeding pot, a disposable paper cup having a diameter of 9 cm was used, one seed was sown per pot, and 121 cups were prepared for each treatment group. The seeded pots were arranged in a greenhouse in a random block arrangement and kept at a humidity of 40 to 60%, and water was periodically supplied as needed. After 4 weeks of cultivation of the pot of the experimental group treated with the microbial agent, 15 ml of mycelia and spore cell culture medium prepared by the above-mentioned shaking culture method were inoculated, and the same amount of water was irrigated with the control pot. In addition, the temperature was maintained at 25 ^o C-32 ^o C and the light used natural light.

After 9 weeks of cultivation, the growth of pepper plants was measured and shown in Table 17.

Treatment Category Pepper seedlings Ground height of pepper seedlings (cm) (average) Untreated control 121 23.7a ^x Powder type microbial delivery media treatment control group 121 24.5a Microbial agent (WYE 20) treatment experimental group 121 27.4b Microbial agent (WYE 324) treatment experimental group 121 28.6b, c

^x Statistically comparing the results of the untreated control and treated experimental groups of the same column

If they differ in the range p = 0.05, they are represented by different alphabets (a, b, c).

As shown in Table 17, the red peppers treated with the microbial agent of the present invention had greatly enhanced growth compared to the untreated control. This indicates that the microbial agent of the present invention is very effective in promoting the growth of plants.

(7) Analysis of microbial agent activity through cultivation of open field pepper

The open field packaging test was carried out on the pepper plants to measure the activity of the microbial agent of the present invention.

The pepper seeds soaked in sterile water for 2 days were immersed in the liquid microbial preparation (I) prepared in Example 3 for 3 hours, and then only the pepper seeds were recovered, and the powdered microbial preparations prepared in Example 5 (particularly experimental group 22) ( 1000 seeds / 4 g). The cell numbers of Streptomyces strains WYE 20 and WYE 324 of the microbial preparation immediately before seed treatment were adjusted to 1.2 × 10 ⁷ cfu / ml and 1.7 × 10 ⁷ cfu / ml, respectively (experimental group). As an untreated control, the seeds which were soaked in sterilized water for 2 hours in sterilized water for 3 hours and then treated with the same delivery medium without the addition of microorganisms were used. Seeds from both experimental and untreated controls were sown in seedlings to provide water periodically and to maintain temperatures between 20 ^° C. and 35 ^° C. When pepper seedlings grow to a size of 1.5 to 2.0 cm, 2.5 g of powdered microbial agent (ie 0.1 g of microbial product per pot) per 25 seedling pots (5 cm x 5 cm, depth 6 cm) After mixing with the soil and filling each pot, red pepper seedlings were transplanted to the mother bed. In the case of untreated control, a mixed soil containing only the same powdered delivery medium without the microbial strain of the present invention was used. It was cultivated in a plastic house where the temperature was maintained at 18 ℃ to 35 ℃ while supplying additional water as needed. The ports are arranged in a random block arrangement. Pepper seedlings of the control and experimental groups were grown in green house for 11 weeks and then planted in open field. Seven days before the establishment of the field, spores and hyphae prepared by the above-described liquid culture method were diluted in water and adjusted to 1.2 to 1.7 x 10 ⁵ cfu / ml, and then irrigated in an amount of 10 ml per port of the experimental group. The control group was irrigated with the same amount of water.

Plant growth and disease occurrence were periodically observed and recorded after the establishment of the field, and the average value and incidence rate (%) are shown in Table 18 (North 1) and Table 19 (North 2).

Treatment Number of plants Plant size (cm) 62 days after planting Plague Incidence (%) 62 days after meal 101 days after meal Untreated control microbial agent (WYE 20) treatment experimental group Microbial agent (WYE 324) treatment experimental group 1,3401,3401,340 78.5a ^x 86.0b90.5c 28.4a ^x 23.5b8.7c 98.6a ^x 89.2b76.1c

^x Statistically comparing the results of the untreated control and treated experimental groups of the same column

If they differ in the range p = 0.05, they are represented by different alphabets (a, b, c).

Treatment Number of plants Plague Incidence (%) 82 days after meal 101 days after meal Untreated control microbial agent (WYE 20) treatment experimental group Microbial agent (WYE 324) treatment experimental group 860860860 26a ^x 0b0b 42.4a ^x 7.6b2.0c

^x Statistically comparing the results of the untreated control and treated experimental groups of the same column

If they differ in the range p = 0.05, they are represented by different alphabets (a, b, c).

As shown in Table 18 (North 1) and Table 19 (North 2), the growth of red pepper crops was greatly promoted and the incidence of late blight was greatly reduced by treatment with the microbial agent of the present invention. This means that the microbial agent of the present invention exhibits very high levels of pepper blight control and growth promoting activity even in actual field field testing.

(8) Analysis of Tobacco Anthrax Control Activity

In order to test the efficacy of WYE 20 and WYE 324, the microbial agent (II) prepared in Example 7, anthrax incidence control activity analysis of tobacco ( Nicotiana tabacum L.) crops in pots was performed.

As a seeding pot, a plastic pot having a diameter of 20 cm was used. One tobacco seedling was planted and grown in each pot containing a potting formulation consisting of agricultural soil and Hotus (UK) (3: 1 by volume). This pot was placed in a random block array in the greenhouse and managed. If necessary, water was supplied and the temperature was maintained at 25 ° C to 30 ° C in natural light.

Microbial agent (II) containing WYE 20 or WYE 324 was diluted 100 times in water (1.0 × 10 ⁴ cfu / ml) and sprayed at weekly intervals before the onset of the disease and the development of anthrax was observed. Tobacco crops in the control group were sprayed with the same amount of water. Tobacco anthrax was caused spontaneously by Colletotrichum nicotianae . The incidence of anthrax was periodically observed, and the presence or absence of anthrax was shown in Table 20.

Treatment Tobacco anthrax outbreak Control + WYE 20 treatment group - WYE 324 treatment group -

+: Anthrax onset. -: Does not develop anthrax. * Anthrax control effect with statistical significance (p = 0.05) between control and treatment.

As shown in Table 20, anthrax did not develop in tobacco crops sprayed with microbial agent (II) containing WYE 20 or WYE 324. On the other hand, anthrax was developed in the tobacco crop of the control group. These results indicate that WYE 20 and WYE 324 are effective in controlling anthrax in tobacco.

(9) Analysis of powdery mildew control activity of Streptomyces spp. WYE 20 (KCTC 0341BP) and Streptomyces spp. WYE 324 (KCTC 0342BP)

The powdery mildew control effect of the liquid microbial preparation (II) prepared according to Example 7 measured the powdery mildew control activity of WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0342BP) of Streptomyces sp.

The microbial agent (II) prepared in Example 7 WYE 20 or WYE 324 was diluted 200-fold (1.0 × 10 ⁴ cfu / ml) was sprayed so that the medicinal solution to the leaves sufficiently. The test packaging was grown in a plastic house, uniformly grown seedlings suitable for planting, and treated with powdery mildew control microorganisms from the beginning of powdery mildew disease. Each microbial agent (II) WYE 20, or WYE 324 was treated with foliar spray at weekly intervals, and the untreated control was sprayed with the same amount of water to ensure the same conditions. The management of the pavement was to provide regular fertilizer and water, which is common practice when cultivating in the farm, so that the crops grew smoothly and the powdery mildew was managed to develop naturally. The treated and untreated groups were treated with three repeated treatments of the ingot method (10㎡ / treatment), treated four times from the beginning of the onset, and investigated the incidence after 7 days. The presence or absence of the damage of microbial agent (II) WYE 20 or WYE 324 Obstruction or condition was visually investigated. Powder powder control results are shown in Table 21.

Treatment Category Pumpkin powdery mild disease Control price (%) 1 repetition 2 repetitions 3 repetitions Average(%) Untreated control 54.9 53.6 46.3 51.6a ^x - Liquid Microbial Actinomycetes WYE 20 (KCTC 0341BP) 18.8 23.0 20.3 20.7b 60 Liquid Microbial Actinomycetes WYE 324 (KCTC 0342BP) 20.5 19.5 24.5 21.5b 58

^{x In the} case of having a statistical significance (Duncan's multiple range test) between the untreated control and the treated column of the same column (p = 0.05), it is represented by different alphabets a and b.

As shown in Table 21, in the case of the pumpkin treated with WYE 20, WYE 324 of the present invention, that is, the microbial agent (II) of the present invention, the onset of pumpkin powdery mildew caused by Sphaerotheca fuliginea was significantly controlled when compared to the untreated control. This indicates that the microbial preparation (II) composed of WYE 20 or WYE 324 of the present invention is very effective in controlling courgette powdery mildew. It was also very effective in the control of powdery mildew against other crops carried out in the same way as in the zucchini described above, and the results are shown in Table 22.

Treatment Category Powder powder control effect package test result Strawberry melon cucumber pepper Lettuce rose Untreated control - - - - - - Liquid Microbial Actinomycetes WYE 20 (KCTC 0341BP) + + + + + + Liquid Microbial Actinomycetes WYE 324 (KCTC 0342BP) + + + + + +

-: Powdery mildew. +: There was a powdery mildew control effect with a statistical significance (Duncan's multiple range test) between the untreated control and the treated group. ※ Powdery mildew pathogen: Strawberry: Sphaerotheca aphanis Melon: Sphaerotheca fusca Cucumber: Sphaerotheca fuliginea Pepper: Leveillula taurica Lettuce: Sphaerotheca sp. Rose: Sphaerotheca panosa .

(10) Analysis of gray mold control activity of Streptomyces spp. WYE 20 (KCTC 0341BP) and Streptomyces spp. WYE 324 (KCTC 0342BP)

The control of gray mold disease of microbial preparation (II) prepared according to Example 7 was measured by the control of strawberry packaging cultivation of gray mold disease control activity of WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0342BP) of Streptomyces spp. It was.

The microbial agent (II) prepared in Example 7 WYE 20 or WYE 324 was diluted 200-fold (1.0 × 10 ⁴ cfu / ml) was sprayed so that the drug solution sufficiently buried in the plant. The test packaging was grown in a plastic house uniformly grown seedlings suitable for planting, and was treated with microbial agents from the beginning of the gray mold. Each microbial agent (II) WYE 20, or WYE 324 was treated by spray spraying at weekly intervals, and the untreated control was sprayed with the same amount of water to ensure the same onset conditions. The management of the pavement provided regular fertilizer and water, which is common in cultivation in general farms, so that the crops grew smoothly and the gray mold disease managed to develop naturally. Treatment and non-treatment were treated with three repeated treatments of the ingot method (10㎡ / treatment), and treated 4 times from the beginning of the onset, and after 7 days, the disease rate was measured and recorded. The condition was visually investigated. The results of gray mold control are shown in Table 23.

Treatment Category Strawberry gray mold disease rate (%) Control price (%) 1 repetition 2 repetitions 3 repetitions Average(%) Untreated control 36.7 43.6 45.4 41.9a ^x - Liquid Microbial Actinomycetes WYE 20 (KCTC 0341BP) 15.5 18.7 19.5 17.9b 57 Liquid Microbial Actinomycetes WYE 324 (KCTC 0342BP) 16.1 15.4 17.7 16.4b 61

^{x In the} case of having statistical significance (Duncan's multiple range test) between the untreated control and the treated column of the same column (p = 0.05), it is represented by different alphabets a and b.

As shown in Table 23, in the case of strawberries treated with WYE 20, WYE 324 of the present invention, that is, liquid microbial agent (II) of the present invention, the incidence of strawberry gray mold caused by Botrytis cinerea was compared with the untreated control. Greatly controlled. This shows that the microbial agent (II) composed of WYE 20 or WYE 324 of the present invention is very effective in controlling strawberry gray mold. It was also very effective in the control of gray fungus disease on other crops carried out in the same way as in the strawberry described above and the results are shown in Table 24.

Treatment Category Gray mold control effect packaging test results pumpkin tomato cucumber pepper chives wave Untreated control - - - - - - Liquid Microbial Actinomycetes WYE 20 (KCTC 0341BP) + + + + + + Liquid Microbial Actinomycetes WYE 324 (KCTC 0342BP) + + + + + +

-: Gray mold disease occurs. +: There is a control effect for gray mold disease with significance (p = 0.05) between the control and the control group (Duncan's multiple range test).

(11) Analysis of Leaf Blight Control Activities of Streptomyces spp. WYE 20 (KCTC 0341BP) and Streptomyces spp. WYE 324 (KCTC 0342BP)

The control effect of the rice leaf blight blight of the microbial preparation (II) prepared according to Example 7 controls the leaf blight control activity of WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0341BP) of Streptomyces sp. Measured.

The microbial agent (II) prepared in Example 7 WYE 20 or WYE 324 was diluted 100-fold (1.0 × 10 ⁵ cfu / ml) and sprayed so that the drug solution to the leaves sufficiently. The test packaging was cultivated by fertilization according to the practice and was treated with microbial agents to control leaf blight from the beginning of the development of leaf blight. Each microbial agent (II) WYE 20, WYE 324 was treated by foliar spray at weekly intervals. Leaf-pattern blight was administered to test packaging to develop naturally. The treated and untreated groups were treated with three repeated treatments of the ingot method (33㎡ / treatment).

Treatment Category Disease rate (%) Vertical vibration (%) 1 repetition 2 repetitions 3 repetitions Average 1 repetition 2 repetitions 3 repetitions Average Untreated control 67.1 95.4 85.5 81.5a ^x 38.0 40.8 37.2 38.2a ^x Liquid Microbial Actinomycetes WYE 20 (KCTC 0341BP) 43 54 50 49b 10.4 13.0 11.1 11.5b Liquid Microbial Actinomycetes WYE 324 (KCTC 0342P) 43 59 53 55b 16.3 10.1 13.2 13.2b

^{x In the} case of having statistical significance (Duncan's multiple range test) between the untreated control and the treated column of the same column (p = 0.05), it is represented by different alphabets a and b.

As shown in Table 25, in the case of rice treated with the WYE 20, WYE 324 of the present invention, that is, the microbial agent (II) of the present invention, the incidence of rice leaf blight blight was significantly controlled when compared to the untreated control. This indicates that the microbial agent composed of WYE 20 or WYE 324 of the present invention is very effective in controlling rice leaf blight.

(12) Analysis of Leaf Blast Control Activity of Streptomyces spp. WYE 20 (KCTC 0341BP) and Streptomyces spp. WYE 324 (KCTC 0342BP) by Rice Cultivation

The control effect of the rice blast control of the microbial preparation (II) prepared according to Example 7 was measured the leaf blast control activity of WYE 20 (KCTC 0341P) and WYE 324 (KCTC 0342P) of Streptomyces genus through rice field cultivation experiment.

The microbial agent (II) prepared in Example 7 WYE 20 or WYE 324 was diluted 100-fold (1.0 × 10 ⁵ cfu / ml) and sprayed so that the drug solution to the leaves sufficiently. The test packaging was grown by direct fertilization of rice seed and fertilized according to the practice, and was treated with microbial agent to control leaf blast from the beginning of seedling blast. Each microbial agent (II) WYE 20, WYE 324 was treated by foliar spray every three days. Leaf blast was administered to the test packaging to occur naturally. The treated and untreated groups were treated with three repeated treatments (1㎡ / treatment) of the ingot, and were treated four times from the beginning of the onset, and the lesion area ratios were examined in Table 26.

Treatment Category Lesion area ratio (%) Control price (%) 1 repetition 2 repetitions 3 repetitions Average Untreated control 34 41 37 37a ^x - Liquid Microbial Actinomycetes WYE 20 (KCTC 0341BP) 15 30 18 21b 43 Liquid Microbial Actinomycetes WYE 324 (KCTC 0342BP) 14 26 11 17b 54

^{x In the} case of having statistical significance (Duncan's multiple range test) between the untreated control and the treated column of the same column (p = 0.05), it is represented by different alphabets a and b.

As shown in Table 26, in the case of rice treated with the WYE 20, WYE 324 of the present invention, that is, the microbial agent (II) of the present invention, the onset of rice leaf blast was significantly controlled when compared with the untreated control. This indicates that the microbial agent composed of WYE 20 or WYE 324 of the present invention is very effective in controlling rice leaf blasts.

(13) Analysis of dollar spot control activity of golf course green grass

The control effect of the microbial agent (II) prepared according to Example 7 on the golf course green grass Bentgrass dollar spot bottle was tested by golf field green field experiments and the dollar spot of WYE 20 (KCTC 0341P) and WYE 324 (KCTC 0342P) of Streptomyces spp. Bottle control activity was measured.

The microbial preparation (II) WYE 20 or WYE 324 prepared in Example 7 was sprayed by 50-fold dilution (1.0 × 10 ⁵ cfu / ml). The untreated control and the treated group were subjected to the ingot method 3 repetition. The area per treatment section was 9 m 2. Treatment was spray sprayed four times at intervals of seven days from the beginning of the onset. The control was sprayed with the same amount of water. The incidence damage area ratio of dollar spots was investigated and shown in Table 27.

Treatment Category % Damage area Control price (%) 1 repetition 2 repetitions 3 repetitions Average(%) Untreated control 18.6 24.2 20.8 21.2a ^x - Liquid microbial pesticides WYE 20 (KCTC 0341BP) 7.3 9.1 7.9 8.1b 62 Liquid microbial pesticides WYE 324 (KCTC 0342BP) 8.1 6.3 6.6 7.0b 67

^{x In the} case of having statistical significance (Duncan's multiple range test) between the untreated control and the treated column of the same column (p = 0.05), it is represented by different alphabets a and b.

As shown in Table 27, when treated with the microbial agent (II) including WYE 20 or WYE 324 of the present invention, the damage area due to the occurrence of dollar spots was greatly reduced. This indicates that the microbial agent of the present invention is very effective in controlling the dollar spot onset of green grass.

The antimicrobial microbial agent of the present invention includes at least one novel antimicrobial strain of Streptomyces spp . WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0342BP), which causes disease in various plants. Excellent control activity of fungal pathogens, including tonia, phytophthora, sclerotenia, piculeria, choletotricum, botrytis, spheroceca, and the like, including plants, especially cucumbers, peppers, grass, pumpkins, The above-mentioned pathogens, including strawberries, melons, lettuce, roses, tomatoes, leeks, green onions, tobacco, and rice, can effectively control diseases caused to various host plants. The antimicrobial microbial agent of the present invention can store the antimicrobial strain in a stable state, and is prepared in powder form and liquid form, mozzarella disease, brown patch, large patch, late blight, powdery mildew, gray mold, anthrax, leaf blight, blast, Dollars It can be effectively used to control a variety of seeding and polio diseases, including pot disease.

Claims (11)

One or more microbial strains selected from the group consisting of Streptomyces spp . WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) for controlling plant growth by controlling fungal pathogens of the plant Microorganisms, including Raiztonia Solani Kuhn, Picureria orisse, Raiztonia cerellis, Sclloretinia homeocapa, Colletotricum graminicola, Colletotricum nicotiane, Bo Antimicrobial microbial agents for use in the control of diseases caused by pathogens such as Tritis cineria and spheroceca. One or more microbial strains selected from the group consisting of Streptomyces spp . WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) for controlling plant growth by controlling fungal pathogens of the plant Antimicrobial microbial agent for use in the control of powdery mildew containing microbial agent. One or more microbial strains selected from the group consisting of Streptomyces spp . WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) for controlling plant growth by controlling fungal pathogens of the plant Antimicrobial microbial agent for use in the control of anthrax, including microbial agent. One or more microbial strains selected from the group consisting of Streptomyces spp . WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) for controlling plant growth by controlling fungal pathogens of the plant An antimicrobial microbial agent for use in the control of a gray fungus comprising a microbial agent. One or more microbial strains selected from the group consisting of Streptomyces spp . WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) for controlling plant growth by controlling fungal pathogens of the plant Antimicrobial microbial agent for use in the control of leaf blight blight comprising a microbial agent. One or more microbial strains selected from the group consisting of Streptomyces spp . WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) for controlling plant growth by controlling fungal pathogens of the plant An antimicrobial microbial agent for use in controlling a microbial agent comprising a microbial agent. One or more microbial strains selected from the group consisting of Streptomyces spp . WYE 20 (KCTC 0341BP) and Streptomyces genus WYE 324 (KCTC 0342BP) for controlling plant growth by controlling fungal pathogens of the plant Antimicrobial microbial agent for use in containing microbial agent for control of dollar spot disease. 8. The antimicrobial microorganism preparation according to any one of claims 1 to 7, wherein the plant is a field crop, a rice crop, a horticulture or a flower crop. 9. The antimicrobial microorganism preparation according to claim 8, wherein the field crop, rice crop, horticulture or flower crop is cucumber, tobacco, pumpkin, strawberry, melon, pepper, lettuce, rose, tomato, leek, green onion, rice or grass. The antimicrobial microorganism preparation according to any one of claims 1 to 7, wherein the microbial delivery medium is a cell culture solution further containing 0.2 wt% of pectin. According to claim 1 to 7, wherein the antimicrobial microbial strain is antimicrobial microbial agent, characterized in that it contains 10 ⁴ to 10 ⁸ as cfu per ml of the microbial delivery medium.