KR101005484B1 - Streptomyces sporoclivatus CJS-49 KCTC 11109BP active against plant fungal pathogens - Google Patents

Abstract

The present invention relates to Streptomyces sporoclibatus CJS-49 having an antimicrobial activity against phytopathogens, and more particularly, the microbial agent of the present invention is an environmentally-friendly microbial agent and the culture medium of the strain is a leaf of a major crop. Treatment with cultivated soil minimizes damage to crops and results in Botritis cinerea ), Pythium ultimum ), Alternaria panax , Fusarium sp., Cylindrocarpon destructans ) and Streptomyces scabies , etc. to effectively control gray mold, spot disease, root rot disease caused by.

Streptomyces sporoclibatus, phytopathogen control agent, microbial pesticide

Description

Streptomyces sporoclivatus CJS-49 KCTC 11109BP active against plant fungal pathogens including Streptomyces sporoclibatus CPS-49 BTCC 11109BP with antimicrobial activity against plant pathogens

The present invention relates to a new strain having antibacterial activity against phytopathogens and microbial preparations comprising the same.

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. These plant pathogenic fungi exist in fungal nuclei and are able to survive in harsh environments such as high and low temperatures, and 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 harsh environmental conditions in diseased tissues or soil and are resistant to degradation and inhibition by neighboring antagonistic microorganisms rather than spores and 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 is not expected to have a great effect, and chemical control has not only significantly reduced the control effect on the packaging due to the recent 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 chemical synthetic pesticides is increasingly regulated due to the toxicity of humans and animals, residual toxicity in soils and crops, and environmental pollution caused by packaging containers. Its use is greatly limited due to the strengthening of specific regulations on pesticides. Therefore, recently, 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 1949s, many studies have been conducted by various 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.

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 , and Fstop (1991, Kodak) using Trichoderma harzianum , Lypodoni of cotton using Trichodex (developed in Israel, 1997) and Kodiak (1990, Gustafson), Pseudomonas fluorescens , seed treatments using the antagonistic bacillus subtilis. Blue Circle, a seed treatment for several crops using DAGGER (1990, Ecogen Inc.), P. cepacia , which controls Ana Pythium sp. (BLUE CIRCLE; 1990, Stine Microbial Products), and Mycostop , a Fusarium wilting disease and parental disease control agent, using Streptomyces griseoviridis with the antagonistic Streptomyces griseoviridis . ). In Korea, research on biological control of ginseng root rot has been undertaken since the early 1970s. In addition to bacteria, the genus Bacillus microorganism into a patent for a fungicide microbial pesticide (Bacillus sp. BS555, KCTC 8821P ), Bacillus poly Rev. (Bacillus polymoxa B36, FERM BP-6068) , Pseudomonas Oreo Pacific Enschede (Pseudomonas aureofaciens B5, FERM BP-6067) , Enterobacter claw dehydratase (Enterobacter cloacae B51, FERM BP-6069) and Bom1 (KFCC 10992) and Bom2 (KFCC 10993) in Streptomyces, an actinomycetes that secrete antifungal substances. In addition, as a microorganism having an antagonistic ability against pathogenic fungi Bacillus subtilis bar amylolytics ( Bacillus subtilis var. amyloliquefaciens KL114, KCTC 8913P), Bacillus amyl Lori query Patience (Bacillus amyloliquefaciens KTGB0202 KCTC 10564BP), Alcaligenes faecalis KL1179, KCTC 8914P), rugi Pseudomonas Ah industrial (Pseudomonas aeruginosa KL1121, KCTC 8915P) , Pseudomonas brother is thiazol car (Pseudomonas aurantiaca KL1326, KCTC 8916P), Bacillus subtilis and Pseudomonas fluorescences, and Pseudomonas fluorescence bios 1 (Biovar 1), which have antagonistic ability against actinomycetes Streptomyces genus, and fungi Del Mar not ahnyum, Penny chamber in Solarium (Penicillium sp.), in Aspergillus (Aspergillus sp.), redundancy parasitic fungi in Acre help monitor the streak lithium BCP (Acremonium strictum, KCTC 0639BP), etc. is 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.

Accordingly, the present invention seeks to provide new environmentally friendly microbial preparations by isolating, identifying and mass culturing new antagonistic microorganisms that exhibit significant antagonistic activity against various plant pathogenic fungi.

The present invention is to search for microorganisms having excellent antagonism against pathogenic microorganisms from all over the country so as to effectively control various pathogenic microorganisms causing various crop diseases, Botrytis Ceria, Pisium Ultimum, It has been invented a new strain having antibacterial activity against various phytopathogenic fungi, such as Alternaria panax, genus Fusarium, Cylindrocarpon destructans.

The present invention antagonist microorganism strain Streptomyces sporoclibatus isolated from soil CJS-49 KCTC 11109BP is the cause of botitis cineraria, which causes gray mold, and pisium and root rot, which cause mozzarella. Not only does it have an excellent inhibitory effect on the genus Fusarium, but it is also active against Streptomyces scavig, the causative agent of potato disease, and Pytopsora capsis, the causative bacterium. It is a very useful invention for the pesticide industry and the environmental industry because it can solve the problem of destruction of ecosystem and killing toxicity.

The present invention provides Streptomyces sporoclibatus CJS-49 [KCTC 11109BP] having antibacterial activity against phytopathogens.

In another aspect, the present invention provides a plant disease control agent containing the mycelia, spores, mixtures or strain cultures of the Streptomyces sporoclibatus CJS-49 [KCTC 11109BP] new strain.

Hereinafter, the present invention will be described in detail.

The present invention is a novel streptocote that effectively controls gray mold, spot, disease, moxa and root rot caused by Botrytis Ceria, Pisium Ultimum, Alternaria Panax, Fusarium, Cylindrocarpon destructans, etc. Myse sporoclibatus CJS-49 KCTC 11109BP The present invention relates to phytopathogen control agents containing mycelia, spores, mixtures or strain cultures thereof as an active ingredient.

In the present invention, the step of separating the microorganism Streptomyces sporoclibatus strains with antagonistic activity such as Botrytis Ceria, Pisium Ultimum, Alterna Panax, Fusarium, Cylindrocarpon destructans, etc. ; After treating the spore suspension of the isolated Streptomyces sporoclibatus strain to agar solid medium, inoculating fungal pathogens and measuring the antibacterial activity to assay the antagonism of the Streptomyces sporoclibatus strain ; Identification of isolated antagonistic Streptomyces sporoclibatus strains; And through the mass culturing step of isolated antagonistic microorganism Streptomyces sporoclibatus strain to prepare a novel microbial agent having antagonistic activity against various phytopathogens.

The novel Streptomyces sporoclibatus CJS-49 according to the present invention is botrytis Ceria, Pisium Ultimum, Alternaria panax, Fusarium genus, silindrocapon in the soil in Naejangsan, Jeongeup-si, Jeollabuk-do Streptomyces sporoclivatus was identified as a result of the selection and isolation of microorganisms having a strong antimicrobial activity common to dextanus . This was named Streptomyces sporoclibatus CJS-49, and was deposited with the Korea Biotechnology Research Institute Gene Bank on April 9, 2007. The accession number is KCTC 11109BP. As a result of analyzing the antimicrobial activity spectrum of the novel Streptomyces sporoclibatus CJS-49 [KCTC 11109BP] of the present invention, Botritis cinerea ), Pythium ultimum ), Alternaria panax ), Fusarium sp., Cylindrocarpon destructans ) and the like has a strong antimicrobial activity. In addition, the culture medium and temperature conditions for the novel Streptomyces sporoclibatus CJS-49 KCTC 11109BP can be effectively produced to have anti-phytopathogenic activity.

Therefore, the present invention includes biopesticides, such as plant disease control agent containing the Streptomyces sporoclibatus CJS-49 KCTC 11109BP culture as an active ingredient.

Such biopesticides may be prepared using a mycelium of the Streptomyces sporoclibatus CJS-49 KCTC 11109BP, a spore, a culture thereof, extracts thereof, and a carrier usable as a pesticide preparation. The carrier may be used as long as it is a commonly used pesticide formulation, but in particular, cryopreservatives such as skimmed milk, yeast extract, PG and electrodeposited SOF49, surfactant TD20A, AS65D, etc. It is preferable. The active ingredient according to the present invention is preferably used in an amount of 0.001 to 90% with respect to the total microbial composition, more preferably by diluting the strain culture extract according to the present invention 100 to 500 times to formulate into a powder. The plant disease control agent thus prepared is preferably treated with a foliage treatment method on the plant's ground from 10 days after planting the crop, or just before the onset, in order to control gray mold, spot pattern disease, mozzarella disease, and root rot disease.

Such microbial preparations may be used as general pesticides such as plant disease control agents, seed coating agents, microbial nutritional agents, soil improving agents and the like.

Hereinafter, the structure of the present invention will be described in detail with reference to Examples. However, the scope of the present invention is not limited by the description of these examples.

Example  1: Isolation and Identification of Strains

1-1. Strain isolate

Soil and compost samples were collected from all over the country to isolate antagonistic microorganisms from Botrytis cieria, Pisium Ultimum and Fusarium. 1 g of the sample was mixed with 10 ml of sterile water, shaken at 28 ° C. for 30 minutes, and diluted to 10 ⁴ times by soil dilution agar plate method. Then, after incubation for 5 days in a 28 ℃ thermostat, a single colony of fungi were separated purely and transferred to GSS slope medium and stored at 4 ℃. Each fungus was selected from Botrytis Ceria, Pisium Ultimum, and Fusarium genus as the test strains, and the best strains showing strong antibacterial activity were selected.

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, and produced a lot of light grayish brown or yellow mycelium depending on the type of medium, and the production of pigment was 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 / white ISP 3 badge Taupe / gray ISP 4 badge Taupe / brown ISP 5 badge Taupe / yellow Spore chain morphology gyre Spore Count / Spore Chain 10-30 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 3.0

+: Possible,-: impossible

I. 16S rDNA Sequencing

In order to confirm the phylogenetic location of the selected strains, 451 bp sequences corresponding to more than 29% of the 16S rDNA sequences were analyzed.

PCR amplification of the 16S rDNA fragment 5'-AGA GTT TGA TCM TGG CTC AG-3 'from the selected strains by primers, and then purified by amplification products and inserted into the TOPO TA cloning kit (Invitrogen), which is E. coli Was transformed. After extracting the plasmid from the transformed E. coli, the nucleotide sequence was analyzed using an Applied Biosystems model 373A automatic sequencer and 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 100767, it was finally confirmed that the strain of the present invention is a Streptomyces sporoclibatus strain. Therefore, on April 9, 2007, it was deposited as a patent strain to the Korea Biotechnology Research Institute Gene Bank and was assigned accession number KCTC 11109BP.

Example  2: various Phytopathogenic bacteria  About Antagonism  black

Phytopathogens include Botrytis , a variety of pathogens that cause significant damage to plants. cineria KACC No.40573, Pythium ultimum KACC No.40495, Alternaria panax KACC No.42141), Fusarium solani (Fusarium solani f. sp . pisi KACC No.42181), Cylindrocarpon destructans KACC No.41077), Phytophtora capsici KACC No.40475), Rhizoctonia solani AG-2-1 KACC No. 40123, and Streptomyces scabis from 8 species of Streptomyces scabis from Korea Agricultural Microbial Resources Center The antimicrobial range was examined by assaying the antagonism of Seth sporoclibatus.

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 ℃, and after the medium is completely hardened, 10μl of the culture medium containing antagonistic microorganisms, 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 3 below. As can be seen in Table 3, as a result of the antagonistic effect of the antagonist microorganism Streptomyces sporoclibatus strain of the present invention and the phytopathogenic bacterium, the strain of the present invention is Botryri which is the cause of gray mold disease It showed excellent inhibitory effect against thymus cineria, Alternaria panax, which causes spots, and Fusarium, which causes root rot, and Cylindrocarpon dextrux. It was confirmed that the phytopsora capsici and the Streptomyces scarbis, the causative agent of potato beetle disease, also had an inhibitory effect.

Test bacteria (fungus) Activity level Botrytis cineria KACC No.40573 ++++ Cylindro car phone destructans
( Cylindrocarpon destructans KACC No.41077) +++ Fusarium solani f. Sp.pisi KACC No.42181) +++ Phytopthora capsici KACC No.40475 +++ Pythium ultimum KACC No.40495 +++ Alternaria panax KACC No.42141 +++ Rhizoctonia solani KACC No. 40123 +++ Streptomyces scabis ) +++ ++++: size of clear zone> 15 mm,
+++: 10-15 mm,
++: 5-10 mm,
+: 1 to 5 mm,
-: No activity

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

After culturing Streptomyces sporoclibatus under the conditions of Example 2 and diluting the culture solution at a constant ratio, Botrytis cine causing caustic fungal 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, which causes Rhi and root rot (Table 4).

Test bacteria
Dilution factor Botrytis Cinema 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 + + 500-fold dilution + - 1000-fold dilution + -

++++: size of clear zone> 15㎜,

+++: 10-15 mm, ++: 5-10 mm, +: 1-5 mm,-: no activity.

Example  4: Streptomyces Sporoclibatus  Culture condition

Antagonist Streptomyces sporoclibatus strains were cultured using a 2.5 L fermenter, and the production medium composition used was starch 5g, glucose 20g, soy flour 25g, juice extract 1g, yeast extract 4g, sodium chloride 2g, potassium phosphate 0.25 g, 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. Culture conditions were incubated for 120 hours at 28 ℃, 150rpm shaking, inoculated 5% (v / v) of the culture medium containing Streptomyces sporoclibatus in 2.5 liter fermenter containing 1.5 liter production medium and at 28 ℃ Incubated for 120 hours under conditions of 200rpm, 1vvm.

1 is a 16S rDNA sequence of the new strain Streptomyces sporoclibatus CJS-49 of the present invention 446bp is analyzed,

Figure 2 is an electron micrograph taken at 10,000 times magnification of the new strain Streptomyces sporoclibatus CJS-49 of the present invention,

Figure 3 shows the antimicrobial activity of the new strain Streptomyces sporoclivatus (CJS-49) of the present invention against Botritis cinerea,

Figure 4 shows the antimicrobial activity of the new strain Streptomyces sporoclivatus CJS-49 of the present invention against Streptomyces scabies ,

Figure 5 shows the antimicrobial activity of the new strain Streptomyces sporoclivatus CJS-49 of the present invention against Fusarium solani ,

Figure 6 shows the antimicrobial activity of the new strain Streptomyces sporoclivatus CJS-49 of the present invention against Cylidrocabon destractans ,

Figure 7 shows the antimicrobial activity of the new strain Streptomyces sporoclivatus CJS-49 of the present invention against Altenarils panax ,

Figure 8 shows the antimicrobial activity of the new strain Streptomyces sporoclivatus CJS-49 of the present invention against Rhizoctonia solani .

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Streptomyces sporoclivatus CJS-49 KCTC11109BP active against          plant fungal pathogens <130> mskim-cjs49 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 451 <212> DNA <213> Streptomyces sporoclivatus <400> 2 ctcttctgca ggtaccgtca ctctcgcttc ttccctgctg aaagaggttt acaacccgaa 60 ggccgtcatc cctcacgcgg cgtcgctgca tcaggctttc gcccattgtg caatattccc 120 cactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc cggtcgccct 180 ctcaggccgg ctacccgtcg tcgccttggt aggccatcac cccaccaaca agctgatagg 240 ccgcgggctc atccttcacc gccggagctt tccacacgga ggtcatgcga ccccgtgtcg 300 tatccggtat tagaccccgt ttccagggct tgtcccagag tgaagggcag attgcccacg 360 tgttactcac ccgttcgcca ctaatcccct cccgaaggag gttcatcgtt cgacttgcat 420 gtgttaagca cgccgccagc gttcgtcctg a 451  

Claims (6)

Botrytis cineria , Pythium ultimum , Alternaria panax , Fusarium solani , Cylindrocarpon destructans , Pytopov Streptomyces sporoclivatus phytopathogenic antagonist strain Streptomyces sporoclivatus showing antimicrobial activity against Phytophtora capsici , Rhizoctonia solani and Streptomyces scabis ) CJS-49 (Accession No. KCTC 11109BP). delete The microbial agent for controlling plant pathogens, comprising the plant pathogen antagonist mycobacterial Streptomyces sporoclivatus CJS-49 (Accession No. KCTC 11109BP) of claim 1. The method of claim 3, A microbial preparation for plant pathogen control, which is prepared by growing and developing Streptomyces sporoclivatus CJS-49 in a medium having an antagonistic ability against a plant pathogen isolated from nature, and diluting it at a dilution of 0 to 200 times. The method of claim 3, The microbial agent for controlling plant pathogens, characterized in that it contains at least one selected from the group consisting of mycelia, spores, and culture of Streptomyces sporoclivatus CJS-49 as an active ingredient. delete

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