KR20080008600A - A novel streptomyces sp. vsv-12 kctc 10936bp active against plant fungal pathogens and preparation of microbial pesticide - Google Patents

Abstract

A novel Streptomyces sp. VSV-12 is provided to be used for biologically and environment-friendly controlling gray mold, root rot and potato cultivars by showing antibacterial activity against plant fungal pathogens. A Streptomyces sp. VSV-12 active against plant fungal pathogens is deposited as a deposition no. KCTC 10936BP. A plant fungal pathogen controlling agent comprises a culture solution of the Streptomyces sp. VSV-12, wherein the pathogen is at least one selected from the group consisting of Botrytis cinerea, Cladosporium fulvum, Fusarium solani, Streptomyces scabiei, Pythium sp., and Phytophthora capsici.

Description

U.S.-12-12CTC1,036XP, which has antibacterial activity against plant pathogens, and a plant pathogen control agent using the same {A novel Streptomyces sp. VSV-12 KCTC 10936BP active against plant fungal pathogens and preparation of microbial pesticide}

1 is a 16S rDNA sequence of the genus Streptomyces sp. 439 bp,

Figure 2 shows the antimicrobial activity of the strain Streptomyces sp. Of the present invention against Botritis cinerea ,

Figure 3 shows the antimicrobial activity of the strain Streptomyces sp. Of the present invention against Cladosporium fulvum ,

Figure 4 shows the antimicrobial activity of the strain Streptomyces sp. Of the present invention against the genus Fusarium sp.

5 is Streptomyces Scar non novel strain Streptomyces genus of the present invention for (Streptomyces scabi) (Streptomyces sp . It shows the antimicrobial activity of).

The present invention relates to Streptomyces sp. VSV-12 having an antimicrobial activity against plant pathogens, and more particularly, the culture or culture filtrate of the strain of the present invention to be treated to the leaves or cultivated soil of the main crop. Asthma, leaf fungus caused by Botrytis cineria , Cladosporium fulvum , Fusarium sp., Streptomyces scabi , etc. The present invention relates to a phytopathogen control agent containing a novel Streptomyces sp. VSV-12 [KCTC 10936BP] mycelia, spores or a mixture thereof as an active ingredient, which can effectively control root rot and potato beetle disease.

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 cineraria, are mostly infectious soils, causing massive damage to various crops. Since these plant pathogenic fungi exist in fungal conditions and survive in harsh environments such as high temperature and low temperature, these fungal nuclei are known to be the primary source of infection for plants every year. Such fungal nuclei are difficult to expect effective control because they can survive in poor environmental conditions in diseased tissues or soil and are resistant to degradation and spores or inhibition by neighboring antagonistic microorganisms than spores or mycelium forms. Methods for controlling such fungi 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 the packaging due to the emergence of drug-resistant pathogens, but also leads to serious environmental pollution by spraying a large amount of chemical pesticides. In addition, the use of chemically synthesized pesticides is increasingly regulated due to toxicity to humans and animals, residual toxicity in soil and crops, and environmental pollution due to packaging containers. Its use is greatly limited due to the strengthening of specific regulations on pesticides. Therefore, in recent years, research on microbiological control has been actively conducted as an alternative method, and the 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 at home and abroad by many researchers. 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. Typical microbial agent developed and commercialized in a foreign country is currently tricot der Do not annuum (Trichoderma harzianum) and tricot der Matthew Polis Forum (T. polysporum) made a BINAB T (1988, ER Butts International ) to inhibit Wood rot fungi, writing GlioGard (1990, WR Grace Co.), a bottle control agent for greenhouse flower seedlings using Gliocladium virens , Fstop (1991, Kodak), Trichodex (1997, Israel) And Pythium sp. Seedlings of cotton using KODIAK (1990, Gustafson) and Pseudomonas fluorescens , which are seed treatment agents using the antagonistic Bacillus subtilis . Seed treatment of various crops using DAGGER (1990, Ecogen Inc.), Pseudomonas Sepacia ( P. cepacia ), which controls Shrop disease, and BLUE CIRCLE (1990, Stine Microbial Products), and Fusarium wilt disease using the antagonist Streptomyces griseoviridis, and MYCOSTOP (1985, Kemira Oy Biocontrol of Finland), a control agent for hairy rot. In Korea, research on biological control of ginseng root rot has been conducted since the early 1970s, and studies on biological control of red pepper disease, cucumber strawberry wilting disease, sesame seedling disease, and ash fungus have been conducted, but the results are insignificant. In addition to the disinfectant microorganism for patented by microbial pesticide bacteria include Bacillus (Bacillus sp. BS555, KCTC 8821P ), Bacillus poly Rev. (Bacillus polymoxa B36, FERM BP- 6068), Pseudomonas Oreo Pacific Enschede (Pseudomonas aureofaciens B5, FERMBP-6067 ), Enterobacter cloacae B51 (FERM BP-6069), and Bom1 (KFCC No. 10992) and Bom2 (KFCC No. 109393) in Streptomyces, actinomycetes that secrete antifungal substances. In addition, Bacillus subtilis var. Amyloliquefaciens KL114, KCTC 8913P, Bacillus amyloliquefaciens KTGB0202 KCTC 10564BP), alkali gin pae Alcaligenes faecalis KL1179, KCTC 8914P), Pseudomonas Pseudomonas aeruginosa KL1121, KCTC 8915P), Pseudomonas aurantiaca KL1326, KCTC 8916P), Bacillus subtilis and Pseudomonas fluorescences, and Pseudomonas fluorescence bios 1 (Biovar 1), which have an antagonistic ability against actinomycetes Streptomyces genus, and fungi are trico Delmar hagianium, Penicillium sp., Aspergillus sp., And Acremonium Strictium BCP, Acremonium strictum (KCTC 0639BP), are being studied.

The principle of biological control by antagonistic microorganisms is to reduce the density or pathogenesis of pathogens through antibiotics produced by antibiotics, parasitic or phagocytosis by cell wall degrading enzymes, and nutrient competition or niche competition. The main mechanisms of action include cross-protection and induction of host resistance using non-pathogenic strains or weakly-pathogenic strains. For example, the inhibitory action of fungal diseases caused by antibiotics or volatile substances, toxins, and special inhibitors secreted by the genus Trichoderma and gliocladium is known. On the other hand, antagonism by parasitic or phagocytosis has been actively studied in recent years. Pathogens caused by chitinase, glucanase, and cellulase, which are cell wall degrading enzymes secreted by antagonistic bacteria Dissolution of is known as the main mechanism of action.

The present invention has been proposed to solve the problems of the prior art as described above, an object of the present invention to provide a VSV-12 genus Streptomyces having antibacterial activity against plant pathogens.

It is another object of the present invention to provide new strains and cultures having antimicrobial activity against phytopathogenic fungi such as Botanical Hospital fungi Botrytis Ceria, Cladosporium full boom, Genus Fusarium, Streptomyces scabies, etc. It is used to control diseases such as disease, leaf fungus, root rot and potato beetle disease, and to provide biological and environmentally friendly drugs and control methods that can control rice blast disease, pepper blight and cucumber ash fungus in the future.

To achieve the above object, the present invention provides a plant pathogen antagonistic strain Streptomyces sp. VSV-12 [KCTC 10936BP].

In another aspect, the present invention provides a plant pathogen control agent comprising a culture solution of the VSV-12 [KCTC 10936BP] genus Streptomyces.

The pathogens of the plant to which the plant pathogen control agent can be applied are Botrytis cineraria, which causes gray mold disease, Cladosporium full boom, which causes leaf fungal disease, and Fusarium solan, which causes root rot disease. , Streptomyces scavy, which causes potato beetle disease, is characterized in that at least one selected from among the causative bacteria of the causative diseases of the wearing and diarrheal diseases, and the phytotopescapa capsaicin, which is the causative agent of the pepper blight.

The present invention is described in more detail below.

The present inventors searched for microorganisms having excellent antagonistic activity against pathogenic microorganisms from soils around the country so as to effectively control various pathogenic microorganisms causing various crop diseases. The present invention has been accomplished by separating, identifying, mass culturing, and formulating a new environmentally-friendly microbial formulation.

Until now, various microbial pesticides having antagonistic activity against plant pathogenic bacteria have been reported, but VSV-12 of the genus Streptomyces with excellent antimicrobial activity of the present invention is a novel microorganism not reported so far for effective control for environmentally friendly agriculture. It is believed to be a formulation.

The present invention is a novel Streptomyces to efficiently control gray mold, leaf fungus, root rot and potato beetle disease caused by Botrytis Ceria, Cladosporium full boom, Streptomyces Skabi in Fusarium Genus VSV-12 [KCTC 10936BP] Provides a phytopathogen control agent containing the mycelia, spores or mixtures thereof as an active ingredient.

The present invention is to isolate the strain from the microorganism Streptomyces genus antagonistic such as Botrytis Ceria, Cladosporium full boom, Fusarium genus and Streptomyces scarby; Treating the spore suspension of the isolated Streptomyces sp. Strain on agar solid medium, inoculating fungal pathogens and measuring the antibacterial activity to assay the antagonism of the Streptomyces spp strain; Identification of isolated antagonistic microorganism Streptomyces spp .; And through the mass culturing step of the isolated antagonistic microorganism Streptomyces genus came to invent a new strain having antibacterial activity against plant pathogens.

VSV-12 genus VSV-12 according to the present invention is selected from the ginseng field soil of Jeollabuk-do and separated and isolated microorganisms having a strong antibacterial activity in common in Botrytis Ceria, Cradosporium full boom and Fusarium and 16S rRNA base As a result of sequencing analysis, it was found to be Streptomyces sp. This was named as VSV-12 in Streptomyces, and was deposited with the Korea Biotechnology Research Institute Gene Bank and deposited accession number KCTC 10936BP on April 18, 2006. As a result of analyzing the antimicrobial activity spectrum of the novel Streptomyces sp. VSV-12 [KCTC 10936BP], antimicrobial activity against Botrytis cieria, Cladosporium full boom, Fusarium spp and Streptomyces scavig It was confirmed. In addition, the culture medium and temperature conditions that can effectively produce novel Streptomyces genus VSV-12 [KCTC 10936BP] to have anti-plant pathogen activity was identified.

 In addition, it is possible to control plant viruses very effectively when the cell culture extract of VSV-12 [KCTC 10936BP] of Streptomyces genus is treated to crops.

Accordingly, the present invention provides a plant disease control agent containing the above VSV-12 [KCTC 10936BP] cell culture solution or culture filtrate of Streptomyces genus as an active ingredient.

The culture filtrate is a supernatant from which the cells are removed from the cell culture medium, and may be obtained by centrifugation or filtration. The culture filtrate may be included in the plant disease control agent in liquid or solid state after drying.

Such plant disease control agents can be prepared by using a carrier which can be used as an agrochemical preparation in addition to the cells of Streptomyces sp. VSV-12 [KCTC 10936BP], its culture solution, its culture solution extract, its culture filtrate. As a carrier, any acceptable carrier may be used as a commonly used pesticide formulation, but in particular, cryopreservatives such as skimmed milk, yeast extract, PG and electrodeposited SOF70, surfactant TD20A, AS65D, etc. It is preferable.

The active ingredient according to the present invention is preferably used 0.001 ~ 90% by weight of the cell culture extract extract with respect to the whole plant disease control agent, more preferably by diluting 0.500 times the strain culture extract according to the present invention is formulated into a powder. . In order to control the plant disease control composition prepared in this way to control gray mold, leaf mold, root rot and Streptomyces Skavy, etc., from 10 days after planting the crop or at the time of onset of the foliage treatment method on the plant ground It is preferable to treat with.

Plant disease control agent of the present invention can be used as a general pesticide, such as plant disease control agent, seed coating agent, microbial nutritional agent, soil improver.

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the contents of the present invention, but the scope of the present invention is not limited to these embodiments.

Example 1 Isolation and Identification of Strains

1-1. Isolation of Strains

In this example, soil and compost samples were collected from all over the country in order to isolate antagonistic microorganisms from Botrytis cieria, Cradosporium full boom and Fusarium genus as test strains. 10 g of the sample was mixed with 10 ml of sterile water, shaken at 28 ° C. for 30 minutes, diluted by 10 ⁴ times with the 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 entire surface. 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 antimicrobial activity common to Botrytis cieria, Cladosporium pullboom and Fusarium genus as test strains.

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 was determined by the medium used. Detailed cultural and physiological characteristics are shown in Table 2 below.

 Analysis item characteristic Spore color / hyphae color by badge type ISP 2 badge Taupe / light yellow ISP 3 badge Taupe / light yellow ISP 4 badge Taupe / light yellow ISP 5 badge Taupe / light yellow Spore chain morphology gyre Spore Count / Spore Chain 10-30 pieces Whether pigments are produced ISP 2 badge U ISP 3 badge U ISP 4 badge radish ISP 5 badge radish 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

I. 16S rDNA Sequencing

In order to confirm the phylogenetic location of the selected strains, 473 bp sequences corresponding to more than 29% of the 16S rDNA sequences were analyzed (SEQ ID NO: 1). 16S rDNA fragment 5'-AGA GTT TGA TCM TGG CTC AG-3 'from the selected strains was amplified by PCR with a primer set, and then the amplification product was purified and inserted into the TOPO TA cloning kit (Invitrogen). Then, it was transformed into E. coli. 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 showed 100% homology with the standard strain Streptomyces genus, it was finally confirmed that the strain of the present invention is Streptomyces genus strain. Thus, on April 18, 2006, it was deposited as a patent strain to the Korea Biotechnology Research Institute Gene Bank and was granted accession number KCTC 10936BP.

Example 2 Antagonistic Assay for Various Plant Pathogens

A plant pathogen is a pathogen Botrytis various germs that cause enormous damage to the plant (Botrytis cineria), leaf mold disease (Cladosporium fulvum), root rot fungus (Fusarium sp.), Potato deodengyi disease (Streptomyces targeted test bacteria of nine kinds of scabi), wearing a bottle-jalrokbyeong (Pythium sp.), pepper Station pathogens (Phytophtora capsici), heukbu pathogens (Alternaria citri), rice blast fungus (Pyricularia oryzae), brown patch (Rhizoctonia solani) The antimicrobial range was examined by assaying the antagonism of the genus New strain Streptomyces. After sterilizing potato agar medium (PDA, Difco), plate and mix 200μl of the culture solution containing mycelia and spores of each pathogen in the medium at 50 ℃. (8 mm in diameter) and then put on a petri dish mixed with pathogens and incubated for 4 days at 28 ℃ to grow various pathogens well. 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 antagonistic microorganism Streptomyces sp. Strain of the present invention and plant pathogens, the strain of the present invention is Botrytis cieria, which causes gray mold disease, It showed excellent inhibitory effect against Cladosporium pool boom, which causes leaf fungal disease, Fusarium, which causes root rot, and Streptomyces scavior, which causes potato beetle disease. In addition, it was confirmed that the phytophthora capsaicin, which is a cayenne pepper disease, also showed an inhibitory effect.

Test bacteria (fungus) Activity level Botrytis cineria +++ Cladosporium fulvum +++ Fusarium sp. +++ Streptomyces scabi +++ 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 activity of Streptomyces genus was re-validated using the agar diffusion method for Botrytis cineria, Cladosporium pullboom, Fusarium genus, and Streptomyces scavior showing good antagonism. Figures 2, 3, 4, 5 are the results showing the activity against Botrytis cieria, Cradosporium full boom, genus Fusarium and Streptomyces scavig in the genus Streptomyces, respectively, Figures 2, 3, 4 and As shown in 5, it was confirmed that it shows very good activity.

Example 3 Antimicrobial Activity According to Dilution Level of Streptomyces spp.

After culturing the genus Streptomyces under the conditions of Example 2 and diluting the culture solution at a constant rate, according to the dilution degree of the culture medium in the same manner as in Example 2, Botrytis cineraria, leaf fungal disease, which causes gray mold disease Antimicrobial activity was measured against Fusarium Solani, which causes crasporium full boom and root rot, which are causes (Table 4).

         Test bacteria dilution factor Botrytis Cinema Sparoceca Macula Fusarium Solani Streptomyces Scarby  Stock solution +++ +++ +++ +++  2-fold dilution +++ +++ +++ +++  5-fold dilution +++ +++ +++ +++  10-fold dilution +++ +++ ++ ++  20-fold dilution ++ +++ ++ +  50-fold dilution ++ ++ + +  100-fold dilution + + - -  200-fold dilution - - - - +++: Clear zone size 10 ~ 15㎜, ++: 5 ~ 10㎜, +: 1 ~ 5㎜,-: No activity

Example 4 Culture Conditions of the Streptomyces spp.

Antagonist microbial strain Streptomyces spp. Was cultured using a 2.5 L fermenter, and the production medium composition 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 2 g of sodium chloride. After dissolving 0.25 g of potassium phosphate and 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 yeast extract, 5 g / l juicy extract and 10 g / l glucose. The culture conditions were shaken at 120 ° C. at 120 ° C. for 120 hours, and 5% (v / v) of the culture medium containing Streptomyces sp. Was inoculated into a 2.5 L fermenter containing 1.5 L production medium, and 200 rpm at 28 ° C. Incubated for 120 hours under the condition of 1 vvm.

As described above, the present invention provides a VSV-12 antagonist microbial strain Streptomyces genus isolated from soil and a plant pathogen control agent using the same, Botrytis cineraria, leaf fungal disease causing gray mold disease Not only does it have an excellent inhibitory effect on Cradosporium full boom, which causes root rot, Fusarium, which causes root rot, and Streptomyces scavior, which causes potato beetle disease, but also on P. It can also be used as an environmentally-friendly, non-polluting biopesticide, as it is also active in the phytophthora capsici, brown patch, which is the red pepper bacterium. As a result, it is possible to solve the problems of destruction of ecosystems and human beings caused by chemical pesticides, which is a very useful invention for pesticide industry and environmental industry.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A novel Streptomyces sp. VSV-12 KCTC 10936BP active against          plant fungal pathogens and preparation of microbial pesticide <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 473 <212> DNA <213> Streptomyces sp. <400> 1 agaactgcgg cgtgcttaca catgcaagtc gaacgatgaa cctccttcgg gaggggatta 60 gtggcgaacg ggtgagtaac acgtgggcaa tctgcccttc actctgggac aagccctgga 120 aacggggtct aataccggat atgacacacg accgcatggt ctgtgtgtgg aaagctccgg 180 cggtgaagga tgagcccgcg gcctatcagc ttgttggtgg ggtgatggcc taccaaggcg 240 acgacgggta gccggcctga gagggcgacc ggccacactg ggactgagac acggcccaga 300 ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgaaagcctg atgcagcgac 360 gccgcgtgag ggatgacggc cttcgggttg taaacctctt tcagcaggga agaagcgaga 420 gtgacggtac ctgcagaaga agcgccggct aactacgtgc cagcagccgc ggt 473  

Claims (3)

Plant pathogen antagonistic strain Streptomyces sp. VSV-12 (KCTC 10936BP). Claim 1 Streptomyces genus VSV-12 (KCTC 10936BP) comprising a culture medium for controlling plant pathogens. The method of claim 2, wherein the pathogen of the plant to which the microbial agent can be applied is Botrytis cieria causing gray mold disease, Cladosporium full boom causing leaf fungal disease, which causes root rot disease. Plant pathogen control, characterized in that at least one selected from Fusarium Solani, Streptomyces scavig that causes potato beetle disease, Pixium, which is the causative agent of stock and disease My.

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