KR100735572B1 - Streptomyces Padanus PSP-32 having antimicrobial activity against major plant pathogens and microbial preparations for controlling plant pathogens using the same - Google Patents

Abstract

The present invention relates to Streptomyces Padanus VSP-32 having an antimicrobial activity against plant pathogens, and more particularly, the microbial agent of the present invention is an environmentally-friendly microbial agent, the culture medium of the strain is a leaf or cultivated soil of the main crop. Treatment to minimize crop damage and reduce Botritis new Streptomyces padanus that efficiently control gray mold, leaf fungus, and root rot caused by cinerea , Cladosporium fulvum , Fusarium sp. VSP-32 [KCTC 10925BP] The present invention relates to a phytopathogen control agent containing mycelia, spores or mixtures thereof as an active ingredient.

Streptomyces Padanus VSP-32, Plant Pathogen Control

Description

A novel Streptomyces padanus VSP-32 KCTC BP active against plant fungal pathogens and preparation of microbial pesticide having antibacterial activity against major plant pathogens

1 is a analysis of 439bp of the 16S rDNA sequence of the new strain Streptomyces padanus of the present invention,

2 is Botrytis cine Ria novel strain of the present invention for (Botrytis cineria) Streptomyces dig Taunus will showing the antimicrobial activity of (Streptomyces padanus),

Figure 3 shows the antimicrobial activity of the new strain Streptomyces padanus of the present invention against Cladosporium fulvum ,

4 is a genus Fusarium sp.) shows the antimicrobial activity of the new strain Streptomyces padanus of the present invention.

The present invention relates to Streptomyces padanus VSP-32 having an antimicrobial activity against plant pathogens, and more particularly, to the culture or the filtrate of the strain of the present invention to be treated to the leaves or cultivated soil of the main crop. Botrytis cineria , Cladosporium fulvum ), Fusarium Plants containing the new Streptomyces Padanus VSP-32 [KCTC BP] mycelium, spores or mixtures thereof as an effective ingredient, which can effectively control gray mold, leaf mold, and root disease caused by sp. It relates to a pathogen control agent.

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, germ-forming germs, such as Botrytis cineria , 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) Developed by KODIAK (1990, Gustafson) and Pseudomonas fluorescens using seed antagonists using Bacillus subtilis and antagonistic bacteria to control cotton lipotonia and Pythium sp. DAGGER (1990, Ecogen Inc.), Pseudomonas Sefar Asia (P. cepacia) for BLUE CIRCLE the seed treatment of multiple crops using (1990, Stine Microbial Products), and antagonistic actinomycetes host Seth sat my very Seo corruption disk (Streptomyces Fusarium wilt with griseoviridis ) and MYCOSTOP (1985, Kemira Oy Biocontrol of Finland), a control agent for hairy ulcer disease. 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, microorganism pesticides are patented microorganisms for bactericides include Bacillus sp. BS555, KCTC 8821P, Bacillus poly Pastor Bacillus Streptomyces spp. , genus Streptomyces, which secrete polymoxa B36, FERM BP-6068), Pseudomonas aureofaciens B5, FERM BP- 6067 , Enterobacter cloacae B51, FERM BP-6069) and antifungal substances Bom1 (KFCC 10992), Bom2 (KFCC 10993), and the like. In addition, as a microorganism having an antagonistic ability against pathogenic fungi Bacillus subtilis bar amylolytic faciens ( Bacillus subtilis var. amyloliquefaciens KL114, KCTC 8913P), Bacillus amyloliquefaciens KTGB0202 KCTC 10564BP), Alcaligenes faecalis KL1179, KCTC 8914P), Pseudomonas aeruginosa (PugPinosa K. Pseudomonas aurantiaca KL1326, KCTC 8916P, Bacillus subtilis and Pseudomonas fluorescences, and Pseudomonas aurantiaca KL1326, Pseudomonas aurantiaca K. The fungi are tricodelma hazinium, Penicillium sp., Aspergillus sp., And the parasitic fungus Acremonium strontium BCP ( Acremonium). strictum, KCTC 0639BP), etc. under study.

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.

Thus, the present invention is a botanical hospital fungi Botrytis Ceria cineria ), Cladosporium fulvum ), Fusarium By providing new strains and cultures with antimicrobial activity against plant pathogenic fungi such as sp.), it is used for the control of gray mold, leaf fungus and root rot, and in the future rice blast, pepper blight and cucumber gray mold It is to provide a biological, environmentally friendly medicine or control method that can control the disease.

In order to achieve the above object, the present inventors searched for microorganisms having excellent antagonism against pathogenic microorganisms from soils around the country so as to effectively control various pathogenic microorganisms causing diseases of various crops. The present invention has been completed by separating, identifying, and culturing a large amount of antagonistic microorganism Streptomyces padanus having excellent inhibitory ability to prepare a new environment-friendly microorganism.

As described above, although various microbial pesticides having antagonistic activity against plant pathogenic bacteria have been reported, Streptomyces Padanus VSP-32 having excellent antimicrobial activity of the present invention is a novel microorganism not reported so far for environmentally friendly agriculture. It is considered to be an effective control agent.

The present invention provides Streptomyces Padanus VSP-32 [KCTC 10925BP] having antibacterial activity against plant pathogens.

The present invention also provides a plant pathogen control agent containing a culture solution or culture filtrate containing the mycelia, spores or mixtures thereof of Streptomyces Padanus VSP-32 [KCTC 10925BP] as an active ingredient.

The present invention is described in more detail below.

The present invention is Botrytis Ceria ( Botrytis) cineria ), Cladosporium fulvum , Fusarium Phytopathogenic fungi containing novel Streptomyces Padanus VSP-32 [KCTC BP] hyphae, spores or mixtures thereof effective in controlling gray mold, leaf fungus and root rot caused by sp. It is about a control agent.

The inventors have found Botrytis cineria ), Cladosporium fulvum , Fusarium sp.) isolating the microbial Streptomyces Padanus strain antagonistic from the soil; Treating the spore suspension of the isolated Streptomyces padanus strain to agar solid medium, inoculating fungal pathogens and measuring the antibacterial activity to assay the antagonism of the Streptomyces Padanus strain; Identification of isolated antagonistic microorganism Streptomyces Padanus strain; And through the mass culturing step of the isolated antagonistic microorganism Streptomyces Padanus strains came to invent a new strain having antimicrobial activity against plant pathogens.

The new strain Streptomyces Padanus VSP-32 according to the present invention is selected from 16 g of microorganisms having a strong antibacterial activity in Botrytis cieria, cradosporium pull boom and fusarium from ginseng field soil in Muan County, Jeollabuk-do, As a result of identification through rRNA sequencing, it was found to be Streptomyces padanus . This was named Streptomyces Padanus VSP-32, and was deposited with the Korea Biotechnology Research Institute Gene Bank on March 20, 2006. The accession number is KCTC 10925BP. As a result of analyzing the antimicrobial activity spectrum of the novel Streptomyces Padanus VSP-32 [KCTC 10925BP] of the present invention, Botritis ( Botritis) cinerea ), Cladosporium fulvum , Fusarium sp.) and the like has a strong antimicrobial activity. In addition, the culture medium and temperature conditions for novel Streptomyces Padanus VSP-32 [KCTC 10925BP] can be effectively produced to have anti-plant pathogen activity.

In addition, it was confirmed that when the extract of Streptomyces Padanus VSP-32 [KCTC 10925BP] cell culture was treated to crops, plant viruses could be effectively controlled.

Accordingly, the present invention provides a microbial preparation for plant disease control containing the Streptomyces Padanus VSP-32 [KCTC 10925BP] cell culture medium or culture filtrate 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 microbial preparation for controlling plant diseases in liquid or solid state after drying.

Such microbial preparations for controlling plant diseases can be prepared using a microorganism of Streptomyces Padanus VSP-32 [KCTC 10925BP], a culture thereof, a culture extract thereof, a culture filtrate, and a carrier usable as an agrochemical preparation. 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 based on the microbial preparation for the control of the whole plant disease, more preferably dilution of the strain culture extract according to the invention 0 to 500 times to formulate into a powder. . In order to control the plant disease control composition prepared in this way to control gray mold, leaf fungus, root rot, etc., it is preferable to treat the plant on the ground of the plant from 10 days after planting or just before the onset of the foliage treatment method. .

Such microbial preparations for controlling plant diseases can be used as general pesticides such as plant disease control agents, seed coating agents, microbial nutritional agents, soil improvers and the like.

Hereinafter, an Example is given and this invention is demonstrated in detail. However, the scope of the present invention is not limited by these Examples.

Example  1: Isolation and Identification of Strains

1-1. Strain isolate

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 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 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 mycelium depending on the type of medium, and the production of the pigment is determined according to the medium used. It shows the characteristics and 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 / 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

In order to confirm the phylogenetic location of the selected strains, 439bp sequences corresponding to more than 27% of 16S rDNA sequences were analyzed. 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). 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 Padanus MITKK-103, so that the strain of the present invention was finally identified as Streptomyces Padanus strain. Thus, on March 20, 2006, it was deposited as a patent strain to the Korea Biotechnology Research Institute Gene Bank and was given accession number KCTC 10925BP.

Example  2: for various plant pathogens Antagonism  black

A plant pathogen is a pathogen Botrytis various germs that cause enormous damage to the plant (Botrytis cineria), leaf mold disease (Cladosporium fulvum ), Fusarium sp., Pythium sp.), Phytophtora capsici , Alternaria citri , Pyricularia oryzae ) and Brown patch (Rhi zoctonia solani ) were tested for the antimicrobial range by antagonizing the new strain Streptomyces Padanus of the present invention. 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) and then placed in a petri dish mixed with pathogens and incubated for 3 to 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 Padanus strain of the present invention and plant pathogens, the strain of the present invention is Botrytis cineraria which causes gray mold disease In addition, it showed excellent inhibitory effects against cladosporium full boom, which causes leaf fungal disease, and fusarium, which causes root rot. It also confirmed that it also showed an inhibitory effect.

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

The activity of Streptomyces Padanus was re-validated using the agar diffusion method for Botrytis cineraria, Cladosporium full boom and Fusarium genus showing excellent antagonism. Figures 2, 3 and 4 show the activity of Streptomyces Padanus in Botrytis cieria, Cradosporium full boom and Fusarium genus, respectively, as shown in Figures 2, 3 and 4 It confirmed that it showed.

Example  3: Streptomyces Padanus  Antimicrobial Activity According to Dilution of Culture Media

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

         Test bacteria dilution factor Botrytis Cinema Cradosporium full boom Fusarium Solani Stock solution +++ +++ +++ 2-fold dilution +++ +++ +++ 5-fold dilution +++ +++ ++ 10-fold dilution +++ +++ ++ 20-fold dilution +++ ++ + 50-fold dilution ++ + + 100-fold dilution + - - 200-fold dilution - - -

+++: size of clear zone 10 ~ 15 mm, ++: 5 ~ 10 mm,

+: 1 ~ 5 mm,-: No activity

Example  4: Streptomyces Padanus  Culture condition

Antagonist microbial strain Streptomyces Padanus was cultured using a 2.5 L fermenter, and the production medium composition was 5 g of starch, 20 g of glucose, 25 g of soy flour, 1 g of juice extract, 4 g of yeast extract, 2 g of sodium chloride, and 0.25 of potassium phosphate. g and 2 g of calcium carbonate were dissolved in 1 L of distilled water, and 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 28 ° C. and 150 rpm for 120 hours, then 5% (v / v) of the culture medium containing Streptomyces padanus 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 conditions of 1vvm.

As described above, the present invention antagonist microorganism mycobacteria strain Streptomyces Padanus VSP-32 KCTC 10925BP isolated from the soil is the crampos cause causative fungal disease, leaf cramps cause crampos Not only does it have an excellent inhibitory effect on the genus Fusarium, which causes fullum and root rot, but it is also active in Pisium, the causative agent of stocking and diarrheal diseases, and the phytophthora capsici brown patch, When used as a non-polluting biopesticide, it is a very useful invention for the pesticide industry and environmental industry because it can solve the destruction of ecosystems and human toxicity.

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Claims (4)

Plant pathogen antagonistic strain Streptomyces padanus VSP-32; Accession number KCTC 10925BP). Streptomyces padanus VSP-32 with antagonism to plant diseases isolated from nature; Microorganism preparation for controlling plant pathogens prepared by growing and developing Accession No. KCTC 10925BP) in a medium and diluting with a dilution factor of 0 to 500 times. According to claim 2, Streptomyces padanus VSP-32 ( Streptomyces padanus ) VSP-32; Microbial agent for controlling plant pathogens, characterized in that the cell containing the Accession No. KCTC 10925BP), culture or a mixture thereof as an active ingredient. According to claim 2, wherein the pathogen of the plant to which the microbial agent can be applied is Botrytis cineraria causing gray mold disease, Cladosporium full boom causing leaf fungal disease, causing root rot disease A microbial agent for controlling plant pathogens, characterized in that at least one selected from Fusarium solani, the causative agent of the stocking and diarrhea, and the phytophthora capsaish, which is the causative agent of red pepper disease.

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