KR100705777B1 - Streptomyces costaricanus vsc-67 kctc 10910bp active against plant fungal pathogens - Google Patents

Abstract

The present invention relates to Streptomyces Costa Ricaus VSC-67 having an antimicrobial activity against phytopathogens , more specifically Botritis cinerea , Rhizoctonia solani ), a new Streptomyces Costa scavenger that effectively controls gray mold, mossola and rice leaf blight, and leaf fungal diseases caused by Cladosporium fulvum and Fusarium sp. ( Streptomyces costaricanus ) VSC-67 [KCTC 10910BP] The present invention relates to phytopathogen control agents containing mycelia, spores or mixtures thereof as an active ingredient.

Streptomyces Costa Ricaus VSC-67, Phytopathogen Control

Description

Streptomyces costaricanus VSC-67 KCTC 10910BP active against plant fungal pathogens with antibacterial activity against plant pathogens

Figure 1 shows the analysis of 450 bp of 16S rDNA nucleotide sequence of Streptomyces Costa Rica.

Figure 2 shows the antimicrobial activity of Streptomyces Costa Rica against Cladosporium fulvum .

Figure 3 shows the antimicrobial activity of Streptomyces Costa Rica against Botrytis cineraria.

Figure 4 shows the antimicrobial activity of Streptomyces Costa Rica against Fusarium sp.

The present invention relates to Streptomyces Costa Ricaus VSC-67 having antimicrobial activity against phytopathogens , and more particularly, Botritis cinerea , Rhizoctonia solani , Cradosporium pulbum (Cladosporium fulvum), Fusarium genus (Fusarium sp.) such as gray mold, damping-off and rice sheath blight, the novel Streptomyces Costa bonus for efficiently controlling the leaf mold disease (Streptomyces costaricanus) that caused by VSC-67 [KCTC 10910BP] The present invention relates to a phytopathogen control agent containing hyphae, spores or a mixture thereof as an active ingredient.

Crop diseases caused by phytopathogenic fungi are a very limiting factor in the stable production of crops along with pests and weeds. Among many phytopathogenic fungi, fungal nuclei, such as Botrytis cineria and Lyattonia solani, are mostly infectious and cause massive damage to various crops. These phytopathogenic fungi exist in fungal nuclei to survive in harsh environments, such as high and low temperatures, so that these fungal nuclei are known to be the primary source of infection for plants every year. It is difficult to expect effective control of these bacteria because they can survive in harsh environmental conditions in diseased tissues or soil, and are resistant to degradation and spores or inhibition by neighboring antagonists than spores or mycelium. The leaf fungus bacterium Cradosporium full boom and the multiply-pattern bacterium Alternaria Solani also cause a great deal of damage to various vegetable crops. Methods for controlling such pathogens include physical methods to improve the cultivation environment, chemical methods using fungicides and microbiological methods using microorganisms. However, physical control alone cannot be expected to have a great effect, and chemical control has not only significantly reduced the control effect on packaging due to the recent emergence of drug resistant bacteria, but also due to the residual toxicity and the residual pesticides caused by the large-scale spraying of chemical pesticides. Due to severe environmental pollution, research on microbiological control as an alternative method has been actively conducted in recent years, and development of effective control methods for environmentally friendly agriculture is urgently required.

As an environmentally safe and effective control method, biological control using a microbial agent is important. Since the early 1970s, many studies have been conducted by many researchers at home and abroad. Recently, more than 10 kinds of microbial products have been commercialized in the world, and research on the development of microbial products is rapidly increasing due to the change of social awareness toward environmentally safe sustainable agriculture.

Representative microbial agents currently developed and commercialized in foreign countries are made of Trichoderma harzianum and T. polysporum to suppress wood decay bacteria (WINAB T, 1988, ER Butts International) GlioGard (1990, WR Grace Co.), a bottle control agent for greenhouse plant seedlings using Gliocladium virens , Fstop (1991, Kodak), Trichodex (1997, Israel) developed) with antagonistic bacteria Bacillus subtilis (Bacillus subtilis), the based seed treatment agent of KODIAK (1990, Gustafson), Pseudomonas fluorescein sense (Pseudomonas fluorescens), Li T. Ana repetition Titanium (Pythium sp.) of the cotton by using the damping-off DAGGER (1990, Ecogen Inc.), Pseudomonas Sefar Asia BLUE CIRCLE (1990, Stine Microbial Products ) of various crops, seed treatment with (P. cepacia) to control the, and the road Actinomycetes Streptomyces and the like so Seo irregularities disk (Streptomyces griseoviridis) the Fusarium (Fusarium) wilt, damping-off control agent MYCOSTOP (1985, Kemira Oy Biocontrol of Finland) using.

In Korea, research on biological control of ginseng root rot has been conducted since the early 1970s, and the research on biological control of pepper blight, cucumber strawberry wilted disease, sesame seedling disease and ash fungus has been conducted continuously. In addition, microorganism pesticides are patented as microbial pesticides, bacteria include Bacillus sp. (Bacillus sp. BS555, KCTC 8821P), Bacillus polymoxa B36, FERM BP-6068, Pseudomonas Pseudomonas aureofaciens B5, FERM BP-6067), Enterobacter cloacae B51, FERM BP-6069, Bom1 (St. KFCC 10992), Bom2 (KFCC 10993) in Streptomyces, an actinomycetes that secrete antifungal substances There is this. Further, the microorganisms having antagonistic ability of the pathogenic fungus of Bacillus subtilis bar amyl duck keupa when Enschede (Bacillus subtilis var. Amyloliquefaciens KL114) [KCTC 8913P], alkali Jeans par faecalis (Alcaligenes faecalis KL1179) [KCTC 8914P ], Pseudomonas Pseudomonas aeruginosa KL1121 [KCTC 8915P], Pseudomonas aurantiaca KL1326 [KCTC 8916P], Bacillus subtilis and Pseudomonas fluorescence lessons, including Streptococcus spp. The fungus has Pseudomonas fluorsons Biobar 1 (Biovar1), and the fungi are trichodelma hazinium, penicillium sp., Aspergillus sp., And the parasitic fungus Acre. Monium Strictium BCP ( Acremonium strictum, KCTC 0639BP) is under study. In order to increase the residual potency of microorganisms in the environment, it is necessary to select and formulate a substrate that can be specifically used by microorganisms, or to treat the substrate itself in advance so that other microorganisms or target pathogens cannot use it. In mass production and formulation of antagonistic microorganisms, it is necessary to select a culture material in consideration of economic feasibility. Antagonistic microorganisms were fungi isolated from domestic soil and showed excellent control effects against Botrytis cineraria, Li foottonia solanie, Cradosporium full boom and Alternaria solani in indoor greenhouse and field experiments.

Conventional microbial preparations using antagonistic microorganisms are known to control phytopathogenic fungi, nematodes and virus diseases, pest control, and broadly weed control. According to these known documents, the principle of biological control by antagonist microorganisms is based on the antibiotics produced by them, the parasitic or phagocytic action of cell wall degrading enzymes, and the density of pathogens through nutrient competition or niche competition. The main mechanisms of action include reducing or reducing pathogenicity, cross-protection and non-pathogenic strains or induction of host resistance. For example, it is known to inhibit fungal diseases caused by antibiotics or volatiles, toxins, and special inhibitors secreted by Trichoderma genus and Gliocladium genus. On the other hand, antagonism by parasitic or phagocytosis has been actively studied recently. Pathogens caused by chitinase, glucanase, and cellulase, which are cell wall degrading enzymes secreted by antagonists Dissolution of is known as the main mechanism of action.

Accordingly, the present inventors have completed the present invention by preparing new environmentally friendly microbial preparations by isolating, identifying, mass culturing and formulating antagonistic microorganisms excellent in inhibiting ability of the phytopathogens for effective biological control of various major plant diseases. Was done.

Accordingly, the present invention provides a new strain and culture medium having antimicrobial activity against phytopathogenic fungi such as Botanical pathogen Botrytis cineria, Lizatonia solani, Cradosporium full boom, etc. The present invention is used to control blight and leaf fungal diseases, and to provide biological, environmentally friendly drugs or methods for controlling rice blasting disease, pepper blight and cucumber ash fungus in the future.

The present invention is characterized by Streptomyces Costa Rica VSC-67 [KCTC 10910BP] having antibacterial activity against phytopathogens.

In addition, the present invention is characterized by another plant disease control agent comprising the Streptomyces Costa Ricaus VSC-67 [KCTC 10910BP] culture.

The present invention will be described in more detail as follows.

The invention Botrytis cine Liao (Botritis cinerea), Li Estonian solani (Rhizoctonia solani), Klein FIG sports Solarium pulbum (Cladosporium fulvum), Fusarium genus (Fusarium sp.) that is endemic or the like gray mold, damping-off and rice sheath blight, the novel Streptomyces to efficiently control the leaf mold disease process Costa Nurse (Streptomyces costaricanus) VSC-67 [ KCTC 10910BP The present invention relates to phytopathogen control agents containing mycelia, spores or mixtures thereof as an active ingredient.

In the present invention, the step of separating the microbial Streptomyces Costa Ricaus strains having an antagonistic activity such as Botrytis cieria, Lizatonia solani, Cradosporium full boom; Treating the spore suspension of the isolated Streptomyces costa Ricanus strain to agar solid medium, inoculating fungal pathogens and measuring the antimicrobial activity to assay the antagonism of the Streptomyces costa Ricanus strain; Identification of isolated antagonistic Streptomyces costa ricas strains; And mass culturing of isolated antagonist Streptomyces Costa Rica strains.

The novel Streptomyces Costa Ricaus VSC-67 according to the present invention is selected and separated from the ginseng field in Muan County, Jeollabuk-do, by selecting microorganisms having a strong antibacterial activity in common to the cladosporium fulvum and Fusarium sp. ㆍ Identified Results, Streptomyces costaricanus ). It was named Streptomyces Costa Ricaus VSC-67 and was deposited with the Korea Biotechnology Research Institute Gene Bank on February 20, 2006. The accession number is KCTC 10910BP. The novel Streptomyces Costa Ricaus VSC-67 [KCTC 10910BP] of the present invention can be applied to Botritis cinerea , Rhizoctonia solani , Cladosporium fulvum , Fusarium genus ( Fusarium sp. In addition, the culture medium and temperature conditions that can effectively produce novel Streptomyces Costa Ricaus VSC-67 [KCTC 10910BP] to have anti-phytopathogenic activity.

In addition, it was confirmed that when the extract of Streptomyces Costa Ricanus VSC-67 [KCTC 10910BP] cell culture was treated to crops, plant viruses could be effectively controlled.

Therefore, the present invention includes biopesticides such as plant disease control agents containing the Streptomyces Costa Ricauss VSC-67 [KCTC 10910BP] culture as an active ingredient.

Such biopesticides may be prepared using Streptomyces Costa Ricaus VSC-67 [KCTC 10910BP] cells, cultures thereof, extracts of the cultures thereof, together with a carrier usable as a pesticide preparation. As a carrier, any acceptable carrier may be used as a conventional pesticide formulation, but in particular, cryopreservatives such as skim milk, yeast extract, PG and electrodeposited SOF70, surfactant TD20A, and AS65D are used. As for the active ingredient according to the present invention, it is preferable to use 0.001 to 90% of the total microbial composition, and more preferably, dilute 100 to 500 times the strain culture solution extract according to the present invention to powder. In order to control the plant disease control agent prepared in this way, in order to control black diseases, gray mold disease, warehousing disease, diarrheal disease, and red pepper germ disease, the leaves are treated on the plant ground from 10 days after planting or just before onset. It is preferable to treat with.

Such microbial agents may be used as general pesticides such as plant disease control agents, seed coating agents, microbial nutritional agents, soil improvement agents.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Isolation and Identification of Strains

1-1. Strain isolate

Soil and compost samples were collected from all over the country in order to isolate the microorganisms with antagonistic resistance to the genus Cradosporium full boom and Fusarium in the present invention. 1 g of the sample was mixed with 10 ml of sterile water and shaken at 28 ° C. for 30 minutes, and then diluted to 10 ⁴ times by soil dilution agar plate method, and 0.1 ml of the diluted solution was dispensed on the GSS medium (Table 1) to evenly spread the whole surface of the medium. Next, after incubation for 5 days in a 28 ℃ thermostat single colony of fungi (single colony) was purely separated and transferred to GSS slope medium and stored at 4 ℃. Each fungus was selected as one of the best strains showing the common antimicrobial activity in the test strains of the genus Cradosporium full boom and Fusarium.

TABLE 1 GSS Badge Composition Table

Badge Ingredient Concentration (g / L) Starch 10 Glucose 20 Soy flour 25 Beef Extract One Yeast extract 4 Sodium chloride 2 Potassium phosphate 0.25 Potassium carbonate 2 pH 7.2

1-2. Strain Identification

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

end. Morphological characteristics

Selected strains were inoculated in GSS agar medium and incubated at 28 ° C. for 5 days, and observed to produce a lot of light grayish brown or yellow mycelia depending on the type of medium, and the production of pigments depends on the medium used. It shows the characteristics to be determined and detailed cultural and physiological properties are shown in Table 2 below.

TABLE 2 Culture Characterization of Streptomyces Costa Rica.

 Analysis item characteristic Spore color / hyphae color by badge type     ISP 2 badge Taupe / yellow     ISP 3 badge Taupe / brown     ISP 4 badge Taupe / yellow     ISP 5 badge Taupe / yellow     Spore chain morphology gyre     Spore Count / Spore Chain 10-50 pieces Whether pigments are produced     ISP 2 badge U     ISP 3 badge radish     ISP 4 badge radish     ISP 5 badge U Carbon use     Arabinose -     Fructose +     Glucose +     Mannitol +     Raffinose -     Sucrose -     Xylose + Whether to grow when the following ingredients are added     5% (w / v) sodium chloride (NaCl) -     100 μg / ml thallium acetate -     1 μg / ml crystal violet +     20 μg / ml streptomycin -     10 μg / ml penicillin -     Lowest pH for growth 3.5

+: Possible,-: impossible

I. 16S rDNA Sequencing

To confirm the phylogenetic location of the selected strains, 450 bp sequences corresponding to more than 29% of the 16S rDNA sequences were analyzed. 16S rDNA fragments from the selected strains were amplified by PCR with primer sets, and then the amplification products were purified and inserted into the TOPO TA cloning kit (Invitrogen), which was transformed into E. coli. After the plasmid was extracted from the transformed E. coli, the nucleotide sequence was analyzed by using an automated base sequence analyzer (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 Costa Rica NRRL B-16897 and Streptomyces Costa Rica KCTC 9879, so that the strain of the present invention was finally confirmed to be Streptomyces Costa Rica. Therefore, on February 20, 2006, it was deposited as a patent strain to the Korea Biotechnology Research Institute Gene Bank and was given accession number KCTC 10910BP.

Example 2 Antagonistic Assay for Various Plant Pathogens

Phytopathogens include a variety of pathogens that cause severe damage to plants, Botrytis cineria , Fusarium sp., And Pythium. sp.), Streptococcus spp., Phytophtora capsici , Alternaria citri , Cladosporium fulvum , Pyricularia oryzae , and Rhi zoctonia solani The antimicrobial range was examined by assaying the antagonism of Mises Costa Rica. After sterilizing potato agar medium (PDA, Difco), plate and mix 200μl of the culture medium containing mycelia and spores of each pathogen in the medium at 50 ℃ (8 mm in diameter) was placed on a petri dish mixed with pathogens and incubated for 3 to 4 days at 28 ° C. where various pathogens were well grown. The effect of antagonistic microorganisms on various pathogens was investigated with a low support diameter, which is shown in Table 3 below. As can be seen in Table 3, as a result of the antagonistic effect of the antagonist microorganism Streptomyces Costa Ricanus strains and phytopathogens of the present invention, the strains of the present invention is responsible for the cause of Fusarium and melanoma It showed an excellent inhibitory effect on the clamsporium full boom, and against Botrytis cineraria, a causative fungus causing caustic fungi and gray fungus, and phytoprosora cappsi brown patch, a causative bacterium. It also confirmed that the inhibitory effect was observed.

Table 3 Streptomyces Costa Rica Antibacterial Range for Pathogenic Fungi

Test bacteria (fungus) Activity level Botrytis cineria +++ Cladosporium fulvum +++ Fusarium sp. +++ Phytopthora capsici + Pythium aphanidermatum - Pythium ultimum - Rhizoctonia solani ++ Pyricularia oryzae -  +++: size of clear zone 10-15 mm, ++: 5-10 mm, +: 1-5 mm,-: no activity

The activities of Streptomyces Costa Ricaus were re-validated using the agar diffusion method for Cladosporium full boom and Fusarium genus showing excellent antagonism. 2 and 3 are the results showing the activities of the genus Cradosporium full boom and Fusarium of Streptomyces Costa Rica, respectively, as shown in Figures 2 and 3 was confirmed to show very good activity.

Example 3 Culture Conditions of Streptomyces Costa Rica

Antagonist microorganism Streptomyces Costa Ricanus strains were cultured using a 2.5L fermenter, and the production medium used at this time was starch 5g, glucose 20g, soybean powder 25g, gravy extract 1g, yeast extract 4g, sodium chloride 2g, potassium phosphate 0.25g, After dissolving 2 g of calcium carbonate in 1 L of distilled water, 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 of yeast extract, 5 g / L of juicy extract, and 10 g / L of glucose. The culture conditions were shaken for 120 hours at 150 ℃ and 28 ℃, 5% (v / v) of the culture medium containing Streptomyces Costa Rica inoculated in a 2.5L fermentation containing 1.5L production medium and 200 rpm at 28 ℃ , 1 vvm incubated for 120 hours.

As described above, the antagonistic microorganism strain Streptomyces Costa Rica KCTC 10910BP of the present invention isolated from the soil is the cause of the cradosporidium full boom causing the black disease, the genus Fusarium causing the wilting disease, and the gray mold disease It is not only an excellent inhibitory effect on Botrytis cineraria, but also active in Pissium, the causative agent of Receiving Disease and Shrop Disease, and the Phytophthora capsici Brown Patch, a caustic bacterium. When used, it is a very useful invention in the pesticide industry and environmental industry because it can solve the destruction of ecosystems and human toxicity.

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Claims (2)

Streptomyces Costa Rica VSC-67 [KCTC 10910BP] with antibacterial activity against phytopathogens. Plant disease control agent comprising a culture medium of Streptomyces Costa Rica VSC-67 [KCTC 10910BP] of claim 1.

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