KR100519469B1 - Use of a bacterium mixture of Burkholderia sp. AB 100 and Pseudomonas sp. AB 62 for preventing ginseng plant pathogens - Google Patents

Abstract

The present invention relates to a microorganism having an antagonistic ability against ginseng pathogens and a microorganism preparation containing the microorganism or its culture as an active ingredient, and more particularly, having a antagonistic ability against ginseng pathogens and having pyrrolnitrin or Burkholderia sp. Strain AB100 or Pseudomonas sp. Strain AB62, which produces 2,4-diacetylphloroglucinol, a mixed strain PB of the two microorganisms -62100 and a microbial agent containing one or two or more of the microorganism and its culture as an active ingredient. The microbial agent containing the microorganism of the present invention can be usefully used as an environmentally friendly microbial pesticide with antagonistic ability against phytopathogenic fungi.

Description

Use of a bacterium mixture of Burkholderia sp. For the use of mixed strains of AA 100 from Berkholdereria and 62 of AJ from Pseudomonas. AB 100 and Pseudomonas sp. AB 62 for preventing ginseng plant pathogens}

The present invention relates to a microorganism having an antagonistic ability against ginseng pathogens and a microorganism preparation containing the microorganism or its culture as an active ingredient, and more particularly, having a antagonistic ability against ginseng pathogens and having pyrrolnitrin or Burkholderia sp. Strain AB100 or Pseudomonas sp. Strain AB62, which produces 2,4-diacetylphloroglucinol, a mixed strain PB of the two microorganisms -62100 and a microbial agent containing one or two or more of the microorganism and its culture as an active ingredient.

Korean ginseng is Korea's unique natural resource, and it is a representative herbal medicine of Korea, which has been widely used for thousands of years as an oriental medicine or medicine for the health of mankind. As Western medicine has shown limitations in overcoming the side effects of synthetic drugs and intractable diseases, the value of herbal medicines and natural products including ginseng is emerging. Ginseng prevents cancer, inhibits aging, protects the liver, strengthens fatigue and improves brain function, and recently, it has been reported to prevent the damage of environmental hormones. Ginseng, the chief of all herbal medicines, is a plant that grows in extremely limited environments. It has a longer growth period than other crops, and its cultivation method is special and difficult, and has been produced only in some countries of the world.

Korea is a ginseng seeding country that produces Korean ginseng with excellent medicinal effects due to its climate and climate suitable for its natural growth and cultivation. However, many countries are paying attention to the ideal conditions that ginseng has as a health food or health drug, and not only existing ginseng producing countries but also non-producing countries are actively promoting policies to encourage the research and production of ginseng. Ginseng growers include Korea, China, Japan, the United States, and parts of Europe. In the past, most of the countries that consumed ginseng were Confucian culture, including Korea, China, Japan, Taiwan and Southeast Asia. This is expanding. Although the ginseng production and consumption market in the world is difficult to collect statistical data accurately due to the characteristics of ginseng distribution structure of each country, diversity of product types and security measures of recent data, it is mainly focused on the import and export of Hong Kong, the center of global ginseng sales market. According to each country's ginseng production statistics data, Korea, which maintained 46% of global ginseng production by the late 80s, decreased its share to around 39% in the 1990s, 0.5% of production growth rate, which is less than the world average growth rate. On the other hand, China, which occupied 43% of the world's production in the 1980s, increased to more than 50% in the 1990s, showing a high annual growth rate of 7.2%. As such, Korean ginseng is weakening its international competitiveness in terms of price, and in order to strengthen its international competitiveness, research on the development of cultivation technology to improve the quality of fresh ginseng is needed. In particular, the most urgent problem in the recent ginseng cultivation is the high incidence rate due to the absolute shortage of proper scheduled land and deterioration of the soil conditions. Therefore, if more pesticides are used, many cases of exports are not possible due to pesticide residues. Intensive research is urgently needed.

Korean ginseng is divided into red ginseng and white ginseng. White ginseng is mainly grown for 4 years. The types of products produced are processed and distributed in the form of fresh ginseng, white ginseng, taeguk ginseng, and fresh ginseng. It is a field where six years of production is produced by contract cultivation with Tobacco Ginseng Corporation, and almost all of them are purchased in the form of fresh ginseng. Regionally, the border between Chungbuk and the red ginseng is predominant in the north and white ginseng in the south.

Ginseng production increased from 1,877 thousand tonnes in 1970 to 4,768 thousand tonnes in 1980 and 15,132 thousand tonnes in 1991, but slightly decreased to 11,971,000 tonnes in 1995. The trend of ginseng production increased by 9.8% per year in the 1970s and 11.1% per year in the 80s. Ginseng production increased significantly since the mid-80s but remained constant in the 1990s.

Many researchers have been concentrating on the development of scientific ginseng cultivation technology, including the establishment of ginseng production base, the improvement and management of seed, improvement of agricultural structure, improvement of cultivation methods and promotion of mechanization. In particular, ginseng is cultivated under the inverted structure unlike other crops, so the variation in quality and yield is large due to environmental factors such as planting location, rainfall, temperature, wind strength, soil fertility, moisture content, breathability, and acidity. The situation is inferior to the safety of crops.

Ginseng is one of the crops that avoids continuous cultivation in the same field due to problems of serial disturbance. Growers say that the field in which the ginseng is grown is reworked. Therefore, ginseng growers have always had difficulty in selecting cropland whenever they planted ginseng, especially in a narrow farmland such as Korea, the situation is very serious enough to reveal a limit to the selection of cultivated land. This problem acts as a significant hazard to domestic and international competitiveness of Korean ginseng, and it is a darkening factor for the future of Korean ginseng cultivation.

Ginseng root rot, which is considered important in ginseng cultivation, has been observed in cropland as well as cropland. Root symptom is thought to be one of the causes of germination and survival rate after cultivation of seedlings. In fact, seedlings planted in farmhouses usually have a residual rate of less than about 50% when harvested after 5 years. This is because root pathogens inhabit the roots of ginseng during herbivore cultivation. In this way, when ginseng is damaged by root germs in cropland, the thick film spores of remaining root pathogens in the soil are distributed evenly in the soil by 7-10 times tillage and rotary work when managing the planned site. If it is more likely to cause damage to ginseng. The root causes of root rot are actually Cylindrocarpon destructans , Fusarium solani , Pythium sp., Phytophthora cactorum , and Erwinia carotobora . ( Erwinia carotovora ) has been reported to be largely involved alone or in combination (Ginseng cultivation, Standard Agricultural Manual p 103, Rural Development Administration, 2000). Root rot is also a problem caused by Rhizoctonia solani , which causes seedlings of seedlings, and Alternaria panax of spots . In order to control these diseases, management of planned sites through the use of organic substances to reduce the density of the pathogens in the soil or to suppress the onset and chemical control using soil fumigants are being attempted. It is becoming. However, it is difficult to achieve a clear effect by the above control methods, and soil infectious pathogens such as ginseng root rot are almost impossible to control because they form a thick spore in the soil. The use of soil fumigants also threatens to prevail in pathogens for five years due to the destruction of the soil ecosystem. In particular, the major soil diseases of ginseng are infected at various temperature ranges, so once they are infected, they will be damaged year-round although the disease will progress slowly. Therefore, for effective soil disease control, it is considered that biological control is most suitable as a countermeasure for continuous ginseng disease that is effective in the nature of ginseng cultivation and is stable for the environment, and shows antagonistic activity against Cylindrocarpon destructans in Korea. The identification and antagonism of Streptomyces species has been reported (Second World Ginseng Symposium, Huh-Sup Chung, Chung-Hee Kim, p 67-74, 1978, Korea Ginseng Research Institute).

Until now, as a means to control the ginseng root rot disease, biological control methods using antagonistic microorganisms and chemical control methods using soil fumigants such as cylon and bassamide, especially in arable lands, and seedling methods such as paddy cultivation of answer Gyunhwan, have been used. The method is not organized. In particular, the biological control studies on ginseng soil disease have been mainly conducted for the selection of antagonistic microorganisms and the evaluation of pavement effect. However, the control effect on ginseng root rot was inconsistent. This phenomenon has been pointed out as a limitation of biological control method using antagonistic microorganisms, and the cause is not only the lack of understanding of various musculoskeletal environment, but also the high level of antimicrobial activity of selected strains against target pathogens, It is considered that the ability of bacteria to settle in soil and seedlings has decreased.

Therefore, the present inventors selected a new useful antagonistic microorganisms for the main ginseng disease in consideration of the cultivation characteristics of pathogens and ginseng for biological control of ginseng soil disease, which is difficult to control, and the selected microorganisms are useful for controlling the ginseng pathogens. The present invention has been completed by revealing that it can be.

An object of the present invention is a novel microorganism Berkholdereria strain AB100, Pseudomonas strain AB62 or a mixed strain of the two microorganisms having an antagonistic ability against ginseng pathogens and one or two or more of the microorganisms and their cultures as an active ingredient. It is to provide a microbial agent containing.

In order to achieve the above object, the present invention provides a novel microorganism Burkholderia sp. Strain AB100, Pseudomonas sp. Strain AB62 and a mixed strain PB-62100 of the two microorganisms.

In addition, the present invention provides a microbial agent for controlling ginseng pathogens containing one or two or more of the microorganism and its culture as an active ingredient.

In addition, the present invention provides a method for producing an antimicrobial material against ginseng pathogens using the microorganism.

Hereinafter, the present invention will be described in detail.

The present invention provides novel microbial Burkholderia sp. Strain AB100, Pseudomonas sp. Strain AB62 and a mixed strain PB-62100 of the two microorganisms.

The microorganism of the present invention is the strain AB100 of the genus Berkholderia which produces pyrrolnitrin represented by the following Chemical Formula 1 and has an antagonistic ability against ginseng pathogens.

In addition, the microorganism of the present invention is Pseudomonas genus strain AB62, which produces 2,4-diacetylphloroglucinol (2,4-diacetylphloroglucinol) represented by the following formula (2) and has an antagonistic ability against ginseng pathogens.

In addition, the mixed strain of the present invention is a mixed strain of the strain Berkholderia strain AB100 and Pseudomonas strain AB62 in a 1: 1 ratio and has an antagonistic ability against ginseng pathogens.

In the present invention, the strains causing the underground disease from the ginseng during the cultivation process was isolated, and novel microorganisms AB100 and AB62 having antagonistic ability against the isolated microorganisms were selected.

The novel strain isolated above had a pale yellow colony for AB 100 and a creamy translucent colony for AB 62 on Tryptic Soy Broth. The morphological characteristics of AB 100 are aerobic bacilli, and the motile species have one to several monopoles. AB 62 is an aerobic bacillus with a size of 0.5 to 1.0 × 1.5 to 5.0 µm, and has one to several genus hairs. The physiological properties of AB 100 and AB 62 are summarized in Table 1 below.

AB 100 AB 62 Gram stain - - Cell diameter (μm) 1.3 0.65 Cell length (μm) 2.35 1.44 No. of flagella > 1 > 1 Fluorescence on King's Medium B - + Grow at 37 ℃ + + Grow at 41 ℃ + + Oxidase reaction - + Gelatin liquefaction - - Starch hydrolysis - - Casein hydrolysis + + Catalase - + Lipase (Tween 80 hydrolysis) + - Reduction of nitrate to nitrite + + Denitrification - - Arginine dihydrolase d + Indole production d d Methyl-red test - - Cellulose Hydrolysis - - Use D-glucose + + Lactose + + Sucrose + + Maltose + + Fructose + + Sorbitol + + Manitol + + β-alanine + + Citrate + + Cellobiose + + Arabinose + +

In addition, as a result of sequencing the 16S rDNA of AB100 isolated above, it has a nucleotide sequence as shown in SEQ ID NO: 1 , which is 99% of 16S rDNA of AB2 of Berkholderia, and 16S rDNA of AB101 of Berkholderia 99%, 99% sequence with the 16S rDNA of Burkholderia Sephacia and 99% sequence homology with 16S rDNA of B. stabilis. Therefore, AB100 isolated above was classified into the genus Burkholderia and named " Burkholderia sp. Strain AB100".

In addition, the nucleotide sequence of 16S rDNA of AB62 isolated above was found to have the nucleotide sequence shown in SEQ ID NO: 2 , which is 97% of P. corrugata's 16S rDNA, and Pseudomonas torassia (P). nucleotide sequence homology of 97% with 16S rDNA of P. fluorescens and 97% with 16S rDNA of P. migulae of P. migulae. Therefore, AB62 isolated above was classified as Pseudomonas genus and named as " Pseudomonas sp. Strain AB62".

In addition, the strain mixed with the strain AB100 genus strain B100 and Pseudomonas genus strain AB62 was named "PB-62100" and was deposited on 21 January 2002 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC). 10162BP).

The microorganism of the present invention produces an antimicrobial agent having an antagonistic ability against ginseng pathogens, Berkholderia strain AB100 produces pyrronitrin, Pseudomonas strain AB62 produces 2,4-diacetylprologusinol do.

In addition, Berkholderia sp. AB100, Pseudomonas sp. AB62, or the mixed strain PB-62100 of the two microorganisms of the present invention, Alternaria panax , Phytophthora cactorum , Lytotonia It has excellent antagonistic ability against Ginseng subterranean pathogens composed of Rhizoctonia solani , Botrytis cinerea and Cylindrocarpon destructans .

In addition, the present invention provides a microbial agent for controlling ginseng pathogens containing one or two or more of the microorganism and its culture as an active ingredient.

The microorganism in the strain is Berkholderia strain AB100, Pseudomonas strain AB62 or a mixed strain thereof PB-62100.

The culture medium of the microorganism includes an antimicrobial substance such as pyrronitrin produced by Berkholderia strain AB100 or 2,4-diacetylprologlucinol produced by Pseudomonas strain AB62.

The present invention provides a microbial agent for controlling Ginseng pathogens containing the pyrronitrin or 2,4-diacetylprologlucinol as an active ingredient. These substances are produced by other antagonists and have been reported to be antimicrobial to several pathogens similar to ginseng pathogens (Karen et al., Applied and Environmental.Microbiology. 1994, 60 (6): 2031-2039; Jos et al. Molecular Plant-Microbe Interaction.1997, 11 (2): 144-152; Jos et al., Phytopathology, 1999, 89 (6): 470-475).

Microorganisms for the control of ginseng pathogens containing one or two or more of the strains of Berkholderia sp. AB100, Pseudomonas sp. AB62, or a mixture thereof, PB-62100 and its culture as an active ingredient of the present invention, stable formulation of the strain It may be prepared in the form of a hydrating, granulating or encapsulating formulation for the purpose.

When formulated using the microorganism or culture ginseng pathogen It may be supplied as contained in the composition for control, or may be stored separately for long-term storage and mixed before use. When the microorganism is supplied separately for long term preservation, the microorganism may be stored at -70 ° C. or lower in glycerol stock solution or may be used by freeze-drying at -20 to -80 ° C.

The hydrating agent of the present invention is prepared by drying and pulverizing a solid medium inoculated with microorganisms, followed by mixing by adding a surfactant and an extender / nutrient.

In the above surfactants, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 At least one selected from the group consisting of one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch.

In addition, the granules of the present invention are prepared by drying and grinding a solid medium inoculated with microorganisms and then adding a surfactant, an extender / nutrient and a disintegrant.

In the above surfactants, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 One or two or more selected from the group consisting of one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch, and bentonite as a disintegrant. (bentonite), talc, dialite, Use De (kaolin), calcium carbonate and at least one or two selected from the group consisting of (calcium carbonate).

Also added to the granules of the present invention are one or more selected from the group consisting of surface active agents, inert carriers, preservatives, wetting agents, feed promoters, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protective agents, buffers and flow agents. It can be prepared by addition.

As a result of replacing the strains of Berkholderia sp. AB100 and Pseudomonas sp. AB62 of the present invention, each strain does not inhibit the antagonism of the other strains, which shows that there is no mutual inhibitory effect between the two microorganisms ( FIG. 1). Reference).

In addition, the microbial agent of the present invention to look at the control effect against several kinds of Ginseng subterranean pathogens, the microbial agent of the present invention exhibits a strong inhibitory action against all underground diseases (see Figure 2 ).

Therefore, the microbial agent of the present invention shows an excellent effect on the control of ginseng subterranean pathogens composed of alternaria panax, phytophthora fetusrum, lysatonia sorani, botrytis cineri and cylindrocarpon distractance.

Treatment of the microbial agent of the present invention is a mixture of the strain AB100 of the genus Berkholdereria and Pseudomonas strain AB62, and then mixed in a ratio of 1: 1 to immerse the seedlings (1 year old) after the cultivation and (1) 0.5 liter (L) was used per each. The microorganism preparations of the present invention were treated with soil collected from Cylindrocarpon distractances and Fujrium genus, which resulted in recognized extreme root rot. As a result, the soil treated with the microbial preparation of the present invention showed more than 74% sterilization power, and the untreated soil showed 78% (22% sterilization power) morbidity.

In addition, the present invention provides a method for producing an antimicrobial material using the microorganism.

Method for producing an antimicrobial material of the present invention

1) culturing Berkholderia spp. AB100, Pseudomonas spp. AB62, or a mixed strain thereof, PB-62100;

2) extracting the culture solution with ethyl acetate and concentrating under reduced pressure; And

3) providing a method for producing pyrronitrin or 2,4-diacetylprologlucinol consisting of the steps of chromatographic separation.

When culturing the strain AB100 of the genus Berkholderia, pyrronitrin is produced, and 2,4-diacetylprologlucinol is produced when the strain Pseudomonas genus AB62 is cultured. In addition, culturing the mixed strain PB-62100 produces pyrronitrin and 2,4-diacetylprologlucinol simultaneously.

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 Identification of Pathogens

The present inventors inoculated the seedlings collected from the soil in sterilized water and then inoculated in agar medium having a size of about 3 mm, and incubated them at 24 ° C. for 3 days to identify them based on their morphological characteristics under a microscope. As a result of separating the causative strains of underground diseases from the ginseng of the cultivation process, the microorganisms are alternaria panax, phytophthora factrum, ly tritonia sorani, botrytis cineri and silindrocapon distractance. It was confirmed that.

<1-1> Isolation of Microorganisms with Antagonistic Capacity against Ginseng Underground Pathogens

The present inventors have isolated a novel microorganism having an antagonistic ability that can inhibit the microorganisms causing the ginseng underground side disease. Specifically, the antagonist strains were collected in a vinyl house plantation of a farmer in Jinju, and after diluting the soil with distilled water several times, the dilution was spread on a yeast-extract agar. Plate was incubated at 28 ° C. for 7 days. Single colonies were screened using the petri plate assay method.

<1-2> Identification of Microorganisms

The present inventors performed physiological and biochemical tests of the strain isolated in Example <1-1> (BERGEY's Manual of Systemic Bacteriology).

As a result, on the Tryptic Soy Broth, AB 100 had pale yellow colonies and AB 62 had creamy translucent colonies. The morphological characteristics of AB 100 were aerobic bacillus, and the motile species had one to several monopoles. AB 62 is an aerobic bacillus with a size of 0.5 to 1.0 × 1.5 to 5.0 µm and had one to several genus hairs. Other physiological properties of AB 100 and AB 62 are summarized in Table 1 .

<1-3> 16S rDNA sequence analysis

The present inventors analyzed the 16S rDNA base sequence of the strain isolated above and compared it with 16S rDNA of other strains.

As a result, the isolated 16S rDNA of AB100 had the nucleotide sequence shown in SEQ ID NO: 1 , which was 99% of 16S rDNA of AB2 of Berkholdereria, 99% of 16S rDNA of AB101 of Berkholderia, and Burke The sequence homology was 99% with 16S rDNA of Holderia Sephacia and 16S rDNA of B. stabilis.

Therefore, AB100 isolated above was classified into the genus Burkholderia and named " Burkholderia sp. Strain AB100".

In addition, the nucleotide sequence of 16S rDNA of AB62 isolated above was found to have the nucleotide sequence shown in SEQ ID NO: 2 , which is 97% of P. corrugata's 16S rDNA, and Pseudomonas torassia (P). nucleotide sequence homology was 97% with 16S rDNA of P. fluorescens and 97% with 16S rDNA of P. fluorescens, and 97% with P. migulae 16S rDNA.

Therefore, AB62 isolated above was classified as Pseudomonas genus and named as " Pseudomonas sp. Strain AB62".

<1-4> Production of Mixed Strains

The present inventors mixed the strain Berkholderia strain AB100 and Pseudomonas strain AB62 isolated above to produce a mixed strain. Specifically, each strain was cultured in a liquid at 28 ° C. using tryptic soy broth as a base medium, and the bacterial cultures were mixed 1: 1 when the bacterial density was 10 ⁸ CFU / ml or more.

The present inventors named the mixed strain "PB-62100" and deposited it on the GenBank of Korea Biotechnology Research Institute on January 28, 2002 (accession number: KCTC 10162BP).

Example 2 Production of Antagonist

The present inventors intended to separate the antagonist in anticipation that the microorganism isolated in Example 1 has an antagonistic ability against the ginseng subterranean pathogen, secrete an antagonist thereto. Specifically, a single colony of each strain was administered to a medium mixed with 1 L of distilled water and 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl, and then cultured at 30 rpm for 24 hours at 140 rpm. 3 ml of seed culture (Seed culture) was transferred to a 3 L flask (Erlenmeyer) and incubated at 30 ° C. for 5 days. The culture solution was centrifuged at 8,000 rpm for 10 minutes to remove the cells, the supernatant was extracted with ethyl acetate and the ethyl acetate layer was concentrated under reduced pressure. This concentrate was separated by flowing chloroform-acetone gradually by silica gel open chromatography. Fractions showing physiological activity were subjected to secondary purification of cyclohexane-ethyl acetate using silica gel open chromatography in 1/0, 100/1, 50/1, 10/1, 1/1, 0 Separation was carried out in the order of / 1. The resulting fractions were developed on a thin layer chromatography (TLC) plate under solvent conditions of cyclohexane-ethyl acetate (4: 1, v / v), and then sprayed with spores grown on potato agar medium on the TLC plate. Incubated for 3-4 days at 28 ℃ and confirmed the antagonist of the antagonist. The antagonist was recovered and the antagonist was purified purely.

Example 3 Structural Analysis of Antagonist

The inventors analyzed the structure of the antagonist purified in Example 2. Specifically, the UV spectrum (Beckman DU-600) analysis of the purified antagonist was performed using methanol as IR Spectra (Bruker IFS 66 spectrophotometer), the sample was a thin film on the KBr window (window) It was obtained using. Sample treatment of ¹ H NMR and ¹³ C NMR spectra (using Bruker AW 500 spectromerter) was prepared by dissolving 25 mg of sample in 0.5 ml of CD ₃ OD and using tetramethylsilane as internal reference. . In addition, mass spectrometry (analysis with the JEOL JMS-DX300 spectrometer) was performed.

As a result, it was confirmed that the antagonists of the present invention are pyrronitrin represented by Formula 1 and 2,4-diacetylprologlucinol represented by Formula 2.

<Formula 1>

<Formula 2>

Example 4 Screening of Biological Activity

The present inventors intended to analyze the physiological activity of the microorganism isolated in Example 1. Specifically, the screening of physiological activity was carried out by mixing the culture medium of strain Berkholderia strain AB100 and Pseudomonas strain AB62 each containing 0 to 200 ppm of antagonist on 1/5 diluted potato agar medium to prepare an experimental medium. 5 mm agar plugs of eggplant pathogens ( Alternaria panax, Phytophthora cactorum, Rhizoctonia solani, Botrytis cinerea, Cylindrocarpon destructans ) were inoculated onto the prepared medium and the growth diameter of the mycelia was measured. Inhibition rate (ED ₅₀ ) was measured using probit analysis. All experiments were performed three times.

As a result, the Pseudomonas genus and Berkholderia spp. Strains of the present invention had antagonistic activity against pathogens associated with five ginseng subterranean diseases. In particular, Pseudomonas strains had stronger antagonism against three strains of Silydrocarpon Destructrence, Phytophthora Factrum and Botrytis cineri than Berkholderia. It showed stronger activity against Alteraria panax, Rhizoctonia solani . As a result, when these two strains are mixed, there are advantages of simultaneously suppressing five pathogens ( Table 2 , Figure 2 ). In addition, as shown in the replacement culture picture of the two strains, each strain did not inhibit the antagonism of the other strains, and did not mutually inhibit the action ( FIG. 1 ).

Low ring (mm)       Pathogen C. Destructiveness P. Factrum A. Panax R. Sorani B. Cineri AB100 10.5 8 11 8 4.5 AB62 13 11 9.5 5.5 6

Preparation Example 1 Preparation of Microbial Preparation

<1-1> Preparation of Hydrating Agent

The present inventors prepared a hydrating powder that is hydrated when diluted in water as a powder for the purpose of stably formulating a strain of PB-62100, a mixed strain of strain Berkholderia strain AB100 and Pseudomonas strain AB62. The hydrating agent was ground by heating, drying the solid medium and adding a surfactant and an extender. That is, the raw powder was dried and pulverized for about 2 hours in a dryer at 70 ° C. Then coarsely crushed powder, surfactant and extender were mixed and mixed evenly. The mixture was ground by passing through a Jet-O-mill (manufactured by Aljet) and the ground powder was tested for hydration to prepare a microbial formulation in the form of a hydrate.

<1-2> Preparation of granules

The present inventors prepared granules which can be used as they are in granular form for the purpose of stably formulating PB-62100 strain which is a mixed strain of strain Berkholderia strain AB100 and Pseudomonas strain AB62. The granules were ground and dried for 2 hours in a dryer at 70 ° C. for pulverization. The pulverized powder was pulverized through a jet-O-mill, and then the microbial pulverized powder, surfactant, extender / nutrient and disintegrant were spores of the strain 50%, polycarboxylate 3%, sodium lignosulfonate 3%, Sodium dialkyl sulfosuccinate 1%, starch 10%, bentonite 20%, talc 13% of the mixture was mixed with about 35% of the total powder of the powder mixture and kneaded. After the dough was finished, it was granulated using a granulator (diameter 1 mm, length 1-5 mm) and dried in a dryer at 70 ° C. The dried product was passed through a 16-30 mesh sieve to remove the powder to prepare a granule.

<1-3> Preparation of Encapsulated Formulations

In order to prepare an encapsulated formulation, the present inventors mixed 11% soy flour, 6% green beans, 4% rice flour, 4% potato starch, 4% rye flour, 3% ocher soil, followed by autoclaving at 121 ° C. for 20 minutes, and then Mix thoroughly again before and cool in cold water. After mixing thoroughly for more than 10 minutes in a stirrer (d = 5cm, 200 rpm or more), while the stirring is continued, add the PB-62100 strain, which is a mixed strain of the strain AB100 in Berkholdereria strain and strain AB62 in Pseudomonas strain, and mix thoroughly A pesticidal encapsulation formulation was prepared.

Experimental Example 1 Port Experiment on the Control Effect of Ginseng Root Disease of Mixed Bacteria

We immersed the mixed strain of Pseudomonas AB62 and Berkholderia AB100 in seedlings, and collected soil of alveolar soil with Cylindrocarpon destructant and mixed it with fresh raw soil at 50:50 for 2 months. 10 seedlings per pot were grown.

As a result, it was found that the ginseng survival rate was significantly higher than that of the control group treated with the mixed bacteria ( FIG. 3 ). The survival rate for each port was shown in Table 3 .

repeat One 2 3 4 5 Average(%) No treatment 20 30 20 20 20 22 Mixed Bacteria Treatment 70 70 50 100 80 74

In other words, the untreated group survived 22%, but the mixed bacteria treated group survived 74%. Statistical results were significant at 1% level as shown in Table 4 .

Sum of squares Degree of freedom Mean squares F-test Tss ¹ 81.6 9 - Rss ² 6.6 4 - Iss ³ 67.6 One 67.6 ** 36.54 (21.20) Ess ⁴ 7.4 4 1.85

¹ Total sum of squares ² Replication sum of squares

³ isolation sum of squares ⁴ error sum of squares

** Significant at 1% level

It was confirmed that the disease-inhibitory effect was more than 1%. In other words, if the reference in the F-test is 21.20% and this shows a significance of 1% or more, the test has a higher significance of 36.54%.

In the present invention, the mixed bacterial solution was cultured individually and mixed at 50:50, and the concentration of Pseudomonas strain at that time was 4 × 10 ⁹ CFU / ml and the concentration of Berkholderia strain was 6 × 10 ¹⁰ CFU /. Ml. The main cause of roots in ginseng packaging is cylindrocarpon distractant, which grows slowly during ginseng cultivation even in the early stages of ginseng (Ginseng field), and rapidly increases after 4 years of root rot. It is known. The mixed bacterium of the present invention shows a high antagonistic ability to suppress pathogens by increasing the survival rate in the alveolar alveolar packaging, from which it can be seen that it will be able to sufficiently suppress the myobacteria gradually increasing in the general packaging. have.

As described above, the strain Berkholderia strain AB100, Pseudomonas strain AB62 or the mixed strain PB-62100 of the two microorganisms of the present invention and its culture have excellent antagonistic ability against ginseng subterranean pathogens, and environmental pollution and human body It is not toxic and can be usefully used as a microbial pesticide for environmentally friendly ginseng underground diseases.

1 is a photograph showing that each strain does not inhibit the antagonism of different strains as a result of replacing the strains of strains AB100 and Pseudomonas strain AB62 of the present invention,

Figure 2 is a mixed strain PB-62100 of the present invention is a phytopathogen Bactorum (A), Laytononia sorani (B), Botrytis cineri (C), Alternaria panax (D) ), A photograph showing analysis of growth inhibition against cylindrocarphone disductance (E) through replacement culture,

Figure 3 is a photograph of the comparison of the seedlings treated with the mixed strain PB-62100 of the present invention compared to the seedlings (control) when no treatment was made when grown in soil contaminated with Cylindrocarpon distractance.

<110> BIORIGIN INC <120> Use of a bacterium mixture of Burkholderia sp. AB100 and Pseudomonas sp. AB62 for preventing ginseng plant pathogens <130> 1p-12-26 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1533 <212> DNA <213> Burkholderia sp. AB100 <400> 1 tatggatccc accttccggt acggctacct tgttacgact tcaccccagt catgaatcct 60 accgtggtga ccgtcctcct tgcggttaga ctagccactt ctggtaaaac ccactcccat 120 ggtgtgacgg gcggtgtgta caagacccgg gaacgtattc accgcggcat gctgatccgc 180 gattactagc gattccagct tcatgcactc gagttgcaga gtgcaatccg gactacgatc 240 ggttttctgg gattagctcc ccctcgcggg ttggcaaccc tctgttccga ccattgtatg 300 acgtgtgaag ccctacccat aagggccatg aggacttgac gtcatcccca ccttcctccg 360 gtttgtcacc ggcagtctcc ttagagtgct cttgcgtagc aactaaggac aagggttgcg 420 ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacagcc atgcagcacc 480 tgcgcgccgg ttctctttcg agcactccca cctctcagca ggattccgac catgtcaagg 540 gtaggtaagg tttttcgcgt tgcatcgaat taatccacat catccaccgc ttgtgcgggt 600 ccccgtcaat tcctttgagt tttaatcttg cgaccgtact ccccaggcgg tcaacttcac 660 gcgttagcta cgttactaag gaaatgaatc cccaacaact agttgacatc gtttagggcg 720 tggactacca gggtatctaa tcctgtttgc tccccacgct ttcgtgcatg agcgtcagta 780 ttggcccagg gggctgcctt cgccatcggt attcctccac atctctacgc atttcactgc 840 tacacgtgga attctacccc cctctgccat actctagcct gccagtcacc aatgcagttc 900 ccaggttgag cccggggatt tcacatcggt cttagcaaac cgcctgcgca cgctttacgc 960 ccagtaattc cgattaacgc tcgcacccta cgtattaccg cggctgctgg cacgtagtta 1020 gccggtgctt attcttccgg taccgtcatc ccccggctat attagaacca aggatttctt 1080 tccggacaaa agtgctttac aacccgaagg ccttcttcac acacgcggca ttgctggatc 1140 aggctttcgc ccattgtcca aaattcccca ctgctgcctc ccgtaggagt ctgggccgtg 1200 tctcagtccc agtgtggctg gtcgtcctct cagaccagct actgatcgtc gccttggtag 1260 gcctttaccc caccaactag ctaatcagcc atcggccaac cctatagcgc gaggcccgaa 1320 ggtccccngc tttcatccgt agatcgtatg cggtattaat gcgcctttcg ccgggctatc 1380 ccccactaca ggacatgttc cgatgtatta ctcacccgtt cgccactcgc caccaggtgc 1440 aagcacccgt gctgccgttc gacttgcatg tgtaaggcat gccgccagcg ttcaatctga 1500 gccaggatca aactctccgt agaaggatcc ata 1533 <210> 2 <211> 312 <212> DNA Pseudomonas sp. AB62 <400> 2 ggcctgggct acacacgtgc tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg 60 agctaatccc acaaaaccga tcgtagtccg gatcgcagtc tgcaactcga ctgcgtgaag 120 tcggaatcgc tagtaatcgc gaatcagaat gtcccggtga atacgttccc gggccttgta 180 cacaccgccc gtcacaccat gggagtgggt tgcaccagaa gtagctagtc taaccttcgg 240 ggggacggtt accacggtgt gatatcatga ctggggtgaa gtcgtaacaa agtagcccgt 300 aggggaacct gc 312

Claims (8)

Burkholderia sp. Strain AB100 which produces pyrrolnitrin represented by the following Chemical Formula 1 and has an antagonistic ability against ginseng pathogens. <Formula 1> Pseudomonas sp. Strain AB62 which produces 2,4-diacetylphloroglucinol represented by the following formula (2) and has an antagonistic ability against ginseng pathogens. <Formula 2> Mixed strain PB-62100 of Accessory strain Burkholderia strain AB100 of claim 1 and Pseudomonas strain AB62 of claim 2 having an antagonistic ability against ginseng pathogens (Accession number: KCTC 10162 BP) A microbial agent for controlling ginseng pathogens, comprising one or two or more of the strains of claim 1 and its culture as an active ingredient. delete The method of claim 4, wherein the ginseng pathogen is Alternaria panax , Phytophthora cactorum , Rhizoctonia solani , Botrytis cinerea , and silin. Microorganisms, characterized in that selected from ginseng subterranean pathogens composed of Cylindrocarpon destructans ( Cylindrocarpon destructans ). A method for controlling ginseng pathogens using one or more of the strains of claim 1 and its culture. 1) culturing Berkholderia spp. AB100, Pseudomonas spp. AB62, or a mixed strain thereof, PB-62100; 2) extracting the culture solution with ethyl acetate and concentrating under reduced pressure; And 3) A method for producing pyrronitrin or 2,4-diacetylprologlucinol, comprising the steps of chromatographic separation.