WO2011005044A2

WO2011005044A2 - Novel strain of streptomyces sporoclivatus which suppresses ginseng root rot

Info

Publication number: WO2011005044A2
Application number: PCT/KR2010/004456
Authority: WO
Inventors: 노문철; 이우송; 박찬선; 김민수
Original assignee: 한국생명공학연구원
Priority date: 2009-07-08
Filing date: 2010-07-08
Publication date: 2011-01-13
Also published as: WO2011005044A3; KR20110004732A; WO2011005044A9; KR101107330B1

Abstract

The present invention relates to a novel strain of Streptomyces sporoclivatus rho-49 having an antagonist action with respect to micro-organic plant pathogens, and more specifically it relates to: a novel strain of Streptomyces sporoclivatus rho-49 having an antagonist action with respect to micro-organisms which cause ginseng root rot; to a microbial preparation for preventing micro-organic plant pathogens, which contains cells or spores or a culture fluid of the strain or a mixture thereof as an active ingredient; and to a production method for the microbial preparation for preventing micro-organic plant pathogens, comprising the step of culturing the Streptomyces sporoclivatus rho-49.

Description

Novel Streptomyces sporoclibatus strain inhibits ginseng root disease

The present invention relates to a novel Streptomyces sporoclivatus rho-49 strain having an antagonistic action against plant pathogens, and in particular, a Streptomyces spore that antagonizes the causative bacteria of ginseng root disease. Culturing step of the microorganism preparation for controlling plant pathogens containing Locclitus rho-49 strain, cells, spores, culture medium or mixtures of the strains as an active ingredient, and the Streptomyces sporoclibatus rho-49 It relates to a method for producing a microbial preparation for plant pathogen control comprising a.

Although there are 7-8 ginseng species in ginseng, the representative one is Korean ginseng, which is currently grown in Northeast Asia. In particular, Korean ginseng is Korea's own varieties, and is a representative herbal medicine of Korea, which has been widely used for thousands of years for oriental medicine as well as for the health of mankind. Ginseng has different morphological characteristics and pharmacological effects depending on the cultivation area, and its use varies considerably. In the case of domestic ginseng, it is known that cancer prevention, anti-aging, liver protection, fatigue recovery, and strengthening brain function, and recently, it has been reported to prevent the damage of environmental hormones.

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. Due to the excellent effect of ginseng, many countries are getting more attention, and not only the existing ginseng producing countries but also non-producing countries are actively implementing 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 ginseng consumer countries were Confucian culture, limited to Korea, China, Japan, Taiwan, and Southeast Asia. Ginseng consumer groups are 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, the diversity of product types and security measures of recent data, According to the ginseng production statistics of each country, Korea, which maintained 46% of the world's ginseng production until the late 80's, decreased its share to around 39% in the 90's. On the other hand, China, which occupied 43% of the world's production in the 80s, increased to more than 50% in the 90s, and has a high annual growth rate of 7.2%. As such, Korean ginseng is weakening its national competitiveness in terms of price, and in order to strengthen its national competitiveness, research on the development of cultivation technology for improving the quality of raw ginseng is urgently needed. In particular, the most urgent problem in the recent ginseng cultivation is because of the limited amount of pesticide residues in ginseng due to excessive use of pesticides due to the high incidence of plant diseases such as ginseng root disease caused by the absolute shortage of proper site and worsening soil conditions. There is an urgent need for intensive research on the safe and large production methods of ginseng-free pesticides.

Many efforts have been made to develop scientific ginseng cultivation techniques, including the establishment of ginseng production base, ginseng seed improvement, management and agricultural structure improvement, cultivation method and promotion of mechanization. In particular, ginseng is cultivated under the inverted structure unlike other crops, so the variation in quality and yield depends on 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. In particular, the biggest problem in ginseng cultivation is the selection of the planned site due to serial disturbances. The site is a land that has not been planted with ginseng, which used to be more than 30 years old, but has been claimed to be more than 7 years since the establishment of the Korea Ginseng and Tobacco Research Institute. The reason for the deadline is that the damage caused by root disease (root rot) and poor growth. Ginseng root rot, which is considered to be 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 as seedlings after planting 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 roots during crop cultivation.

In this way, when ginseng is damaged by root pathogens in cropland, when the ginseng is grown as a series, the thick film spores of remaining root pathogens in the soil are distributed evenly in the soil by 7-10 times tiller and rotary operation 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, It has been reported that the disease caused by Erwinia carotovora, etc. is largely involved alone or in combination (Ginseng cultivation, Standard Agricultural Manual p 103, Rural Development Administration, 2000). In addition, root rot is a problem caused by Rhizoctonia solani, which causes seedlings of seedlings, and Alteraria panax of spots.

In order to control such diseases, the 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 the chemical control using the soil fumigant are being attempted. It is carried out. However, it is difficult to achieve a clear effect by the above control method, and soil infectious pathogens, such as ginseng root rot, form thick film spores in the soil, so it is very difficult to completely control the long term even in poor conditions. In addition, the use of soil fumigant also removes harmful microorganisms and useful microorganisms at the source, there is also a risk of destruction of the soil ecosystem. In addition, as a way to control the ginseng root rot diseases, biological control methods using antagonistic microorganisms, and especially in the case of arable land, chemical control methods using soil fumigants such as cylon and bassamide, and seedling methods such as paddy cultivation of answering hwanhwan, have been used. The method is not yet organized. Above all, the biological control research on ginseng soil disease has been mainly conducted on the selection of antagonistic microorganisms and the evaluation of pavement effect, but the control effect on ginseng root rot has not been consistent. It is pointed out as a limit.

Therefore, the present inventors have studied the microbiological control as an environmentally friendly ginseng root disease control measures to improve the fundamental structure of the soil in consideration of the cultivation characteristics of pathogens and ginseng for biological control of difficult to control ginseng soil disease. As a result of intensive efforts to carry out the results, a new antagonistic microorganism was selected for the main ginseng root disease, the microorganism confirmed the excellent antagonistic effect against plant pathogens including ginseng and completed the present invention.

Still another object of the present invention is a method for producing a microbial agent for controlling plant pathogens, comprising the step of growing and developing a Streptomyces sporoclibatus rho-49 strain having a antagonistic ability against a plant pathogen isolated from nature in a medium. To provide.

An object of the present invention is to provide a strain of Streptomyces sporoclivatus rho-49 KCTC 11525BP having an antagonistic action against plant pathogens.

Another object of the present invention is to provide a microbial agent for controlling plant pathogens, which comprises cells, spores, culture medium or mixtures of Streptomyces sporoclibatus rho-49 as an active ingredient.

Strain Streptomyces sporoclibatus rho-49 of the present invention has an excellent inhibitory effect against the genus Psirium, which is the cause of ginseng root disease, Cylindrocarpon destructans, and Mozalok disease. In addition, it is also very useful for the pesticide industry and the environmental industry because it can be used as an environmentally friendly, non-polluting biopesticide, as it is active in the phytophthora pactorum, a causative agent of red pepper disease. .

1 is a 16S rDNA nucleotide sequence of the new strain Streptomyces sporoclibatus of the present invention Figure 450bp analysis.

2 is an electron micrograph taken at 20,000 times magnification of Streptomyces sporoclibatus.

3 is a diagram showing the antimicrobial activity of the new strain Streptomyces sporoclibatus of the present invention against Cyclodrocabon destractans .

Figure 4 is a diagram showing the antimicrobial activity of the new strain Streptomyces sporoclibatus of the present invention against Pythium ultimum .

Figure 5 is a view showing the antimicrobial activity of the new strain Streptomyces sporoclibatus of the present invention against Botritis cinerea .

Figure 6 is a diagram showing the antimicrobial activity of the new strain Streptomyces sporoclibatus of the present invention against Fusarium solani (Fusarium solani).

7 is a diagram showing the antimicrobial activity of the new strain Streptomyces sporoclibatus of the present invention against Altenarils panax .

Figure 8 is a view showing the antimicrobial activity of the new strain Streptomyces sporoclibatus of the present invention against Rhizoctonia solani .

9 is a graph showing the growth curve of the new strain Streptomyces sporoclibatus with incubation time.

10 is a graph showing the Streptomyces sporoclibatus antagonistic effect on Cylindrocarpon destructans.

As one aspect for achieving the object of the present invention, the present invention relates to a Streptomyces sp. Strain having an antagonistic action against plant pathogens, and more particularly to plant pathogens, in particular ginseng myedema It relates to a novel Streptomyces sporoclivatus rho-49 strain having antagonism against the resulting plant pathogen.

As used herein, the term "Streptomyces sp." Is a representative genus of soil actinomycetes, which produces antibiotics, and is a fungus belonging to the actinomycetes Streptomyces family. Iii) collectively collectively having the characteristic of forming a conidia in the chain. Preferably, the novel Streptomyces sporoclibatus according to the present invention is isolated from the soil having good parts, and selected and separated as a result of identifying and identifying microorganisms having strong antibacterial activity in common in the phytopsora pactrum and collecticum aquatum. Streptomyces sporoclivatus has been identified. The inventor named it Streptomyces sporoclibatus rho-49, and deposited it on July 06, 2009 at Korea Institute of Bioscience and Biotechnology, and was given accession number KCTC 11525BP.

The strain of the present invention can be used without limitation to various plant diseases caused by plant pathogens, preferably can be used for ginseng root disease.

The term "ginseng root disease" in the present invention refers to root rot disease in other words, since various causes are involved alone or in combination, the symptoms vary greatly, and black or brown color is indicated depending on the state of corruption. Initially, black or brown spots are formed by typical bacteria, but as the disease progresses, various symptoms are caused by nematodes and other bacteria.

Examples of pathogens that cause ginseng root disease include Cylindrocarpon destructans, Rhizoctonia solani, Botrytis cinerea, Fusarium solani, and blood Tophthora cactorum and Pythium ultimum, but are not limited thereto. In addition, it is most often caused by a complex infection that is expressed in correlation rather than being solely caused by each of these pathogens.

In addition, the strain of the present invention is also effective in various plant diseases caused by the pathogen. Examples of the diseases caused by the pathogens include Botrytis cineraria, which causes gray mold, Fusarium solani, which causes root rot, Pytopusora, which is a causative agent of stock and mozzarella diseases Mentorum and the like, the type of disease caused by the plant pathogen is not limited thereto.

Preferably, the strain of the present invention has a strong antagonism against the pathogens of various kinds of plant pathogens causing ginseng root disease, more preferably has a strong antagonism against the cylinder caractone dextans (Fig. 3 And FIG. 10).

From a preferred embodiment, the Streptomyces sporoclibatus rho-49 strain of the present invention has an antagonistic action against plant pathogens, and to confirm this, the antimicrobial activity of the strain against various plant pathogens was analyzed. It was confirmed that there is a strong antimicrobial activity against the pathogen causing ginseng root disease, Cylindrocarpon destructans, the complex pathogen Fusarium sorani, Pisium genus and phytophthora totorum (Fig. 3 to 8). In particular, it was confirmed that the Streptomyces sporoclibatus rho-49 strain of the present invention is a very good antagonist against Cylindrocarpon destructans, the main cause of ginseng root disease (FIG. 10). In addition, Streptomyces sporoclibatus rho-49 confirmed the antimicrobial activity against Altenarils panax , which causes spots disease (FIG. 7).

As such, the strain of the present invention exhibits strong antagonism against pathogens causing various plant diseases including ginseng root disease, and when the strain is used as an environmentally friendly, pollution-free biopesticide, various plant diseases including ginseng root disease can be prevented. It is useful for solving ecosystem destruction and toxicity by artificial medicine.

In another embodiment, the present invention provides a Streptomyces sporoclibatus rho-

The present invention relates to a microbial preparation for plant pathogen control using 44 strains, and preferably to a microbial preparation for plant pathogen control comprising the strain cell, spores, culture solution or a mixture thereof of the present invention as an active ingredient.

The microbial agent of the present invention comprises a biopesticide such as a plant disease control agent containing Streptomyces sporoclibatus rho-49 strain cells, spores, culture medium or mixtures thereof, which have an antagonistic action against plant pathogens, as an active ingredient. It may include. The culture medium included in the microbial preparation of the present invention is a supernatant from which the cells are removed from the cell culture medium, which can be obtained by centrifugation or filtration, and can be included in the microbial preparation for controlling plant pathogens in a liquid or solid state after drying. Such a microbial preparation may be prepared using a carrier which can be used as an agrochemical preparation in addition to Streptomyces sporoclibatus rho-49 strain, its culture solution, its culture extract, and its culture filtrate. As a carrier, any acceptable carrier may be used in a conventionally used pesticide formulation, but the antifungal compound of the present invention may be prepared by a method known in the pharmaceutical field for use as a pesticide, and by itself or pharmaceutical It can be prepared and used in various formulations by mixing with an acceptable carrier (forming agent), diluent (diluent) and the like. In addition, the formulation may use a coating flocculant to adhere the antifungal active bacteria well to the plant leaves or to decompose well in the soil.

From a preferred embodiment, the microbial preparation of the present invention was found to have a very high antagonism and antimicrobial activity against plant pathogens causing ginseng root disease (Examples 2 and 4), the streptomyces spoklori of the present invention Microbial preparations, including Batus rho-49 strains, spores, cultures or mixtures thereof, may be usefully used to inhibit ginseng root disease.

Preferably, the active ingredient according to the present invention is to use the cell culture extract in an amount of 0.001 to 90% with respect to the entire microbial composition, more preferably, dilute the strain culture extract according to the present invention 100 to 500 times to formulate it as a powder. Let's do it. The microbial preparations for controlling plant pathogens prepared as described above are plant roots after planting crops to control ginseng root disease, ash fungus, root rot disease, wearing disease, mozzarella disease, red pepper disease and pathogens causing the plant disease. It is preferable to process by the foliage treatment method in the process. Such microbial agents can be used as general pesticides such as plant disease control agents such as ginseng root disease, seed coating agents, microbial nutritional agents, soil improving agents.

As another aspect, the invention microorganisms for controlling plant pathogens, comprising the step of growing in culture medium Streptomyces sporoclibatus rho-49 strain having antagonism against plant pathogens isolated from nature It relates to a process for the preparation of the formulation.

Preferably, the present invention comprises the step of growing and developing in the medium a plant pathogen isolated from nature, that is, Streptomyces sporoclibatus rho-49 strain having an antagonistic activity against ginseng root disease-induced pathogens, It relates to a method for producing a microbial agent. In more detail, the preparation method of the microbial preparation of the present invention may be applied to plant pathogens such as Cylindrocarpon dextanans, Lizatonia solani, Botrytis cineria, Purarium solani, Phytopsora chitorum and fishium Ultimum. Separating the microorganism Streptomyces sporoclibatus strain antagonistic against the soil; Identification of isolated antagonistic Streptomyces sporoclibatus strains; After treating the spore suspension of the isolated Streptomyces sporoclibatus strain to agar solid medium, inoculating fungal pathogens and measuring the antimicrobial activity to assay the antagonism of the Streptomyces sporoclibatus strain step; And mass production of the isolated antagonist Streptomyces sporoclibatus strains (Example 1).

Microbial preparations for controlling plant pathogens according to the present invention are ginseng root disease-inducing pathogens, for example, Cylindrocarpon destructans, Rhizoctonia solani, Botrytis cinerea ), Fusarium solani, Phytophthora cactorum, Pythium ultimum, but are not limited thereto.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

실시예 1: 균주의 분리 및 동정Example 1 Isolation and Identification of Strains

1-1: Strain Separation

In this experiment, soil samples were collected from all over the country to isolate microorganisms having antibacterial activity against Cylindrocarbon destructans , which causes ginseng root disease, as a test strain. Sampling was conducted by selecting a depth of about 15 centimeters of the topsoil layer and removing foreign matter such as weeds and leaves. It was sealed in a 200 ml glass bottle and then transported to the laboratory at 0-4 ° C. 1 g of the collected sample was mixed with 10 ml of sterile water, shaken at 28 ° C. for 30 minutes, and diluted to 10 ^4-6 times by soil dilution agar plate method. Then, 0.1 ml of the diluted solution was dispensed into YM medium and evenly spread over the whole surface of the medium, followed by incubation for 2 to 4 days in a 30 ° C. incubator, and the resulting colonies were purely separated and transferred to a YM solid medium for storage at 4 ° C. In addition, each fungus as a test strain was analyzed for the antimicrobial spectrum using 8 different species, including Phytoprosora Cactorum.

1-2: Strain Identification

Identification of the strains selected above was carried out as follows.

end. Morphological characteristics

Selected strains were shown in Table 1 by observing the culture and physiological characteristics of a single colony obtained by incubating in YM agar medium for 3 days at 30 ℃ culture in various media as shown in Table 1.

Table 1

I. 16S rDNA Sequencing

In order to confirm the phylogenetic location of the selected strains, 446 bp sequences corresponding to more than 29% of the 16S rDNA sequences were analyzed. PCR amplification of 16S rDNA segment 5'-AGA GTT TGA TCM TGG CTC AG-3 'from the selected strains with a primer set, and then amplified product was purified and inserted into the TOPO TA cloning kit (Invitrogen). E. coli was transformed. After the plasmid was extracted from the transformed E. coli, the nucleotide sequence was analyzed using an Applied Biosystems model 373A automatic sequencer and a kit (BigDye Terminator Cycle Sequencing kit, Perkin-Elmer Applied Biosystems). 16S rDNA of the selected strains were analyzed for homology using the gene bank blast program (Fig. 1). As a result, the selected strain exhibited 100% homology with Streptomyces sporoclibatus NBRC 13847, so that the strain of the present invention was finally confirmed to be a Streptomyces sporoclibatus strain. Therefore, on July 6, 2009, it was deposited with the Korea Institute of Bioscience and Biotechnology, located at 111, Gwahak-ro, Yuseong-gu, Daejeon, Korea, and was given accession number KCTC 11525BP.

실시예 2: 각종 식물병원균에 대한 길항력 검정Example 2: Antagonistic Assay for Various Phytopathogens

Cylindrocarpon destructans KACC 41077 Streptomyces scabies , Pythium utimum KACC 40705 (root disease), Colletotrichum acutatum KACC 40042 (anthrax disease), Botrytis cinerea KACC 40573 (rayworm), Phytophthora cactorum KACC 40166 (root disease, late blight), Rhizoctonia solani AG-2-2 (IV) KACC 40123 (Mozalococcal disease) were obtained from eight specimens from the Korea Center for Agricultural Microbiology and Streptomyces sporoclibatus Drag was tested to determine the antimicrobial range. After sterilizing potato agar medium (PDA, Difco), plate and mix 200 μl of the culture medium containing mycelia and spores of each pathogen in a medium at 50 ° C. (8 mm in diameter) and placed on a petri dish mixed with pathogens and incubated at 28 ° C. for 3-4 days to grow various pathogens. The effect of antagonistic microorganisms on various pathogens was investigated with a low support diameter, which is shown in Table 2 below. As can be seen in Table 2, as a result of the antagonistic effect of the antagonistic microorganism Streptomyces sporoclibatus strain of the present invention and the phytopathogen, the strain of the present invention is a cylinder that is the main pathogen of ginseng root disease It was confirmed that there is a strong antimicrobial activity against carpon destructans and the combined causes of fusarium sorani, fishium, phytophthora puttorum. In addition, it was confirmed that the bacterium causing anthrax, colletotricum akutoom, and altenaria panax, which causes spots, also exhibited an inhibitory effect.

TABLE 2

It showed excellent antagonism in most test strains, and this variety of antimicrobial activity suggests the possibility of application to various crops with one product. In addition, in order to re-verify the activity of Streptomyces sporoclibatus, very good antibacterial activity was confirmed for each test bacterium showing antimicrobial activity (FIGS. 3 to 8).

실시예 3: 스트렙토마이세스 스포로클리바투스 대량 배양Example 3: Streptomyces Sporoclibatus Mass Culture

Antagonist microorganism Streptomyces sporoclibatus strains were cultivated using a 2.5 L fermenter, and the production medium used was 5 g of starch, 20 g of glucose, 25 g of soy flour, 1 g of juice extract, 4 g of yeast extract, and sodium chloride. 2 g, 0.25 g of potassium phosphate, and 2 g of calcium carbonate were dissolved in 1 L of distilled water, and then a conventional liquid medium having a pH of 7.2 was prepared and used. Species culture was incubated in a 500 ml Erlenmeyer flask using a 100 ml liquid medium containing 5 g / L yeast extract, 5 g / L juicy extract and 10 g / L glucose. The culture conditions were shaken for 120 hours at 28 ℃ and 150 rpm, and then 5% (v / v) of the culture medium containing Streptomyces sporoclibatus was inoculated into a 2.5 L fermenter containing 1.5 L of production medium and 28 ℃. Incubated for 120 hours at 150 rpm, 1 vvm condition (Fig. 9).

실시예 4: 스트렙토마이세스 스포로클리바투스의 길항 효과Example 4 Antagonistic Effect of Streptomyces Sporoclibatus

Mix Cylindrocarpon Destrotans and Streptomyces Sporoclibatus in a ratio of 100: 1 to 1:10, and then spray them into the soil, respectively, and then streptomyces sporocli to Cylindrocarpon Destructans. The degree of growth inhibition of Batus was analyzed. As shown in FIG. 10, the ratio of Cylindrocarpon destractans and Streptomyces sporoclibatus shows an excellent inhibitory effect even at 50: 1. It was confirmed that it is a very good antagonist against the main causative bacillus cylinderlox destructans.

Claims

A strain of Streptomyces sporoclivatus rho-49 KCTC 11525BP that has an antagonistic action against plant pathogens.
The method of claim 1, wherein the plant pathogens are Cylindrocarpon destructans, Lyzoctonia solani, Botrytis cinerea, Furarium solani, Blood Strain selected from the group consisting of Phytophthora cactorum and Pythium ultimum.
The strain according to claim 2, wherein the botrytis cineria is a causative agent of ginseng root disease and gray mold.
The strain according to claim 2, wherein the furarium solanie is a causative agent of ginseng root disease and root rot disease.
The strain according to claim 2, wherein the pisidium Ultimum is a causative agent of ginseng root disease, wearing disease and mozaloccosis.
The strain according to claim 2, wherein the phytophthora nytorum is a causative agent of ginseng root disease and red pepper disease.
A microbial agent for controlling plant pathogens, comprising cells, spores, culture medium or mixtures of the Streptomyces sporoclibatus rho-49 strain of any one of claims 1 to 6 as an active ingredient.
A method for producing a microorganism preparation for controlling plant pathogens according to claim 7, comprising the step of growing and developing the Streptomyces sporoclibatus rho-49 of any one of claims 1 to 6 in a medium.
The method of claim 8, wherein the plant pathogens are Cylindrocarpon destructans, Lyzoctonia solani, Botrytis cinerea, Furarium solani, Blood A method for producing a microbial agent for controlling plant pathogens, which is selected from the group consisting of Phytophthora cactorum and Pythium ultimum.