KR100604203B1 - -46 Streptomyces cheonanensis sp. nov. VC-A46 strain effective against plant pathogens and fungicides using the same - Google Patents

Abstract

The present invention is a plant disease using a novel Streptomyces cheonanensis sp.nov.VC -A46 strain isolated from soil having antimicrobial activity against phytopathogens and this strain or a culture filtrate of the strain. It relates to a fungicide for control. According to the present invention of the above-described configuration, Phytophthora capsici , Colletotrichum orbiculare , Magnaporthe grisia , Fusarium oxysporum , It is effective in controlling plant diseases caused by Alternaria mali and Rhizoctonia solani .

Phytopathogens, Streptomyces, Fungicides

Description

Novel Streptomyces Cheonan Ensis VC-A46 strain having antibacterial activity against phytopathogens and fungicides for controlling plant diseases using the same [Streptomyces cheonanensis sp. nov. VC-A46 strain effective against plant pathogens and fungicides using the same}

1 is a scanning electron micrograph of the Streptomyces Cheonanensis VC-A46 strain according to the present invention.

Figure 2 is a schematic diagram based on the 16S rDNA nucleotide sequence of 30 Streptomyces Cheonan Ensis VC-A46 strain according to the present invention.

Figure 3 is a photograph showing the antimicrobial effect against phytopathogens of Streptomyces Cheonanensis VC-A46 strain according to the present invention.

Figure 4 is a view showing the 16S rDNA sequence of Streptomyces Cheonanensis VC-A46 strain according to the present invention.

The present invention relates to a novel Streptomyces cheonanensis sp. Nov. Strain having antibacterial activity against phytopathogens , fungicides and plant control methods using the same.

In general, cucumber anthrax, rice fever, and pepper blight are very important plant diseases with significant economic damage. Oyster anthrax and rice fever are severely responsible for a significant reduction in yield in the entire crop harvest.

In order to control this, cultivation or rotation of resistant varieties is currently performed, but until now, the most effective control method is known to be chemical control using a fungicide. However, due to the increasing use of organic synthetic pesticides, adverse effects on the environment and human beings such as pesticide residues in the soil have reached severe levels, causing social problems.

From this point of view, it is urgently needed to develop a low-toxic, pollution-free fungicide that can be used to control plant diseases such as cucumber anthrax, rice fever, and red pepper disease. In particular, the use of secondary metabolites secreted from microorganisms as a low-toxic pollution-free fungicide is proposed, because the microbial secondary metabolite has a number of useful properties as a fungicide for controlling plant diseases.

In other words, since antibiotics derived from microorganisms are biosynthesized in the natural state, they are rapidly degraded in the natural environment and have little damage due to soil residual toxicity, and due to various chemical structures and biological effects, It has the advantage of reducing side effects, including the appearance, damage to livestock.

The genus Streptomyces, on the other hand, was discovered in 1943 by Waksman and Henry, and has since become widely known as a useful microorganism for the industry of producing antibiotics. This important Streptomyces genus contains a large number of species, and the search for useful Streptomyces species is ongoing.

It is an object of the present invention to provide a novel Streptomyces strain having antimicrobial activity against phytopathogens.

Another object of the present invention is to provide a low-toxic and environmentally friendly fungicide that can be used for plant disease control using the novel Streptomyces strain.

Still another object of the present invention is to provide a method of controlling plant diseases using the novel Streptomycesses strain.

In order to achieve the above object, a novel Streptomyces cheonanensis sp. Nov. VC-A46 strain was isolated from soil and the antimicrobial activity was confirmed to complete the present invention.

Therefore, the present invention provides a novel Streptomyces cheonanensis sp.nov . VC-A46 strain having antibacterial activity against phytopathogens .

In order to achieve another object of the present invention, the present invention provides an environmentally friendly fungicide that is low toxicity and does not cause adverse effects on the environment that can be used for controlling the plant diseases including the novel Streptomyces strain or the culture filtrate of the strain. .

The present invention also provides a method for controlling plant diseases using the novel Streptomycesses strain or the culture filtrate of the strain.

Hereinafter, a novel Streptomyces cheonanensis sp. Nov. Strain having antibacterial activity against phytopathogens according to the present invention configured as described above, a fungicide using the same and a method for controlling plant diseases It demonstrates in detail, referring.

The present inventors separated about 1,000 actinomycetes from soil samples in various regions of Korea and tested the disease control effect on agar medium. As a result, new actinomycetes having excellent antibacterial effect against plant diseases including cucumber anthrax, rice fever, and red pepper disease VC-A46 strain was selected.

Actinomycetes VC-A46 strain according to the present invention was identified as a new species through morphological characteristics, physiological and biochemical characteristics, and 16S rDNA sequence analysis, Streptomyces cheonanensis VC-A46 ( Streptomyces cheonanensis sp. Nov. VC-A46) and then deposited [Microbial Accession Number: KCCM 10641P].

More specifically, Streptomyces cheonanensis VC-A46 strain according to the present invention generates substrate hyphae on the medium, diaminopimelic acid (DAP) of the cell wall is L-form (L-form), the result of observing the spore shape with an electron scanning microscope, it is a reflexible (rectiflexible) type, characterized in that the spore surface is smooth (see Fig. 1).

In addition, the Streptomyces strain VC-A46 showed an important characteristic different from other actinomycetes as G + C content of 75.5%, which is important for bacterial identification, and fatty acid analysis revealed that C _{16: 0} iso, C _{16: 0} and C _{16. : 1} cis 9 dominates (see Table 2).

Referring to Table 2, the Streptomyces strain VC-A46 is a melanin pigment in ISP 2, 3, 5, 7 medium It breaks down escurin, gelatin, arbutin and starch, but does not break down tyrosine and cellulose.

The strain is L-Arabinose (Arabinose), Adonitol, Dextran (Dextran), D-Fructose, Mannitol (Mannitol), Meso- Injitol (meso- inositol, D-Melezitose, D-Melibiose, Raffinose, L-Rhamnose, Sucrose, Xylitol, Xylose ) Can be used as a carbon source.

The strain also contains L-Cystein, L-Valine, L-phenylalanine, L-histidine, amino-n-butyric acid and L- Hydroxylproline can be used.

Furthermore, 16S rDNA nucleotide sequence analysis of the Streptomyces Cheonansis VC-A46 strain according to the present invention (SEQ ID NO: 1, Figure 4). Based on this, a schematic diagram (FIG. 2) was prepared and registered in Genbank (AY822606).

On the other hand, Streptomyces Cheonanensis VC-A46 strain according to the present invention are Phytophthora capsici , Phytophthora capsici , Colletotrichum orbiculare , Magnaporthe grisia , Fusarium oxysporum , It has antimicrobial activity against apples, Alternaria mali , Rhizoctonia solani , etc. (see Fig. 3).

The isolated Streptomyces Cheonanensis VC-A46 strain and the culture filtrate of the strain according to the present invention are all naturally derived materials, which are easily decomposed by microorganisms in the natural environment. Therefore, the strain and the culture filtrate according to the present invention can be prepared in a pollution-free, environment-friendly fungicide composition by mixing a filler or the like commonly used in the art to which the present invention belongs, it can be very useful for controlling plant diseases. .

Hereinafter, the present invention configured as described above will be described in more detail with reference to the following examples. These examples are intended to illustrate the invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Selection of Antagonistic Actinomycetes

In order to select antagonistic actinomycetes, soil 10cm deep from topsoil was collected from vegetable garden soil in Cheonan, mixed 5g of sample soil with 50ml of sterile water, and shaken for 30 minutes with a rotary shaker (150rpm). Prepared.

The prepared suspension was filtered through Whatman 1 filter paper and diluted with sterile water in a 1: 100 (v: v) ratio. 1 ml of each dilution was dissolved at 40 ° C. Humic acid vitamin agar: 1.0 g humic acid, 0.5 g Na ₂ HPO ₄ , 1.71 g KCl, 0.05 g MgSO ₄ · 7H ₂ O, 0.01 g FeSO _{4 · 7H 2 O, 0.02g CaCO} 3, 0.5mg thiamine-HCl, 0.5mg riboflavin, 0.5mg niacin, 0.5mg pyridoxin-HCl, 0.5mg inositol, 0.5mg Ca-pantothenate, 0.5mg p-aminobenzoic acid, 0.25mg biotin, 50mg cycloheximide, 18g agar, 1 liter H ₂ O, adjusted to pH 7.2; vitamins and cycloheximide were filter-sterilized and malt extract agar: 3g malt extract, 0.5g peptone , 0.5g glucose, 0.85% NaCl, 18g agar, 1 liter H ₂ O), and the inoculated agar plate medium was incubated at 28 ° C for 14 days until colonies were generated.

Among the floras thus produced, the floras that appeared to be actinomycetes were selected. Here, the bacterium that appears to be actinomycetes refers to a flora that forms a rigid mycelium on an agar plate by forming a substrate mycelia unlike ordinary bacteria.

Phytophthora capsici , Colletotrichum orbiculare , Magnaporthe grisia , Fusarium oxysporum , Alta The antimicrobial activity against Alternaria mali and Rhizoctonia solani was tested.

Specifically, the selected actinomycetes are streaked at 30 mm away from the plate of V8 juice agar (V8A) and incubated at 27 ° C. for 36 hours, and then placed in the center of the medium. Mycelial disks (mycelial disk, 7 mm in diameter) removed from the cultures of phytopathogenic fungi, such as the growing Oythrax aneurysm ( Colletotrichum orbiculare ), and cultured again for 5 days and confirmed the generation of low support (see FIG. 3).

The antagonistic bacteria selected by the production of low support were grown in YMB liquid medium and mixed with 15% glycerol and stored at -70 ° C.

Example 2 Identification of Actinomycetes Species Using Morphology, Physiology, Biochemical Properties and 16S rDNA Sequence Analysis of Antagonistic Actinomycetes VC-A46

To identify strain VC-A46 of the present invention isolated and selected in Example 1, morphological, physiological and biochemical characterizations were first conducted. Physiological and biochemical tests for species identification were carried out using the methods described in the Manual's manual of systematic bacteriology.

Reference strains for, S. thermolineatus KCCM 41146 ^T (= DSM41451 ^T ), S. macrosporus 15 KCCM 41250 ^T (= DSM41449 ^T ), S. cattleya KCCM 11825 ^T (= JCM4925 ^T ), and S. acidiscabies 16 KCCM 41240 ^T (= ATCC49003 ^T ) It was used from the preservation center (KCCM). Strain VC-A46 and the reference strains of the present invention were incubated at yeast / malt agar medium (4 g yeast extract, 10 g malt extract, 4 g glucose, 1 L water), 28 ℃, mixed with 15% glycerol It was stored at -70 ° C.

The morphology, physiology and biochemical characteristics of strain VC-A46 of the present invention analyzed as a result of the test are summarized in Table 1. Strain VC-A46 of the present invention generates substrate hyphae on the medium, diaminopimelic acid (DAP) of the cell wall is L-form (L-form), and spores were observed by electron scanning microscope. The result was a reflexible (rectiflexible) type, and the spore surface was found to be smooth, indicating that the spleptomyces lie (see FIG. 1).

VC-A46 strain of the present invention is a melanin pigment in ISP 2, 3, 5, 7 medium Aesculin, gelatin, arbutin, and starch were degraded, but not tyrosine and cellulose.

In addition, the strain is L-arabinose (Arabinose), adonitol (Adonitol), dextran (Dextran), D-fructose, Mannitol (Mannitol), meso- inotitol (meso -inositol, D-Melezitose, D-Melibiose, Raffinose, L-Rhamnose, Sucrose, Xylitol, Xylose Xylose) could be used as a carbon source.

The strain also contains L-Cystein, L-Valine, L-phenylalanine, L-histidine, amino-n-butyric acid and L- Hydroxylproline could be used.

Characteristic (Characteristic) VC-A46 S.thermolineatus ^T S. cattleya ^T S.macrosporus ^T Spore chain * RF RF SP RF Aerial hypae + + +-Melanin production +---Max NaCl tolerance (%, w / v) 7% 6% 3% 6% Spore mass color / substrate mycelium color on ISP medium 2 ^a PY / PY DG OP None ISP medium 3 G / O---ISP medium 4 GW / PY---ISP medium 5 O / O-OW-ISP medium 6 GW / VY-None-ISP medium 7 GW / W-OW-Soluble pigment ISP medium 3 DG-None-ISP medium 5 O-None-ISP medium 6 None-SB-ISP medium 7 BW-None-Degradation activity Esculin +--+ Gelatin + + + + Starch + +- + Tyrosine-+ +- Arbutin + + + + Cellulose - - - - Resistance to antibiotics Neomycin (50 ㎍ / ml) - - - - Oleomycin (100 ㎍ / ml) - - - - Penicillin G (10 iu / ml) + + + + Lifampicin (50 ㎍ / ml) - - - - Growth temperature 4 ℃ - - - - 45 ℃ - + + + Growth on pH 5--+-11-+ + + Growth on sole nitrogen source (0.1%, w / v) DL-α-Amino-n-butyric acid + + + + L-Cysteine + + + + L-Histidine + + + + L-Valine + + + + Growth on sole carbon source (1%, w / v) Adonitol + + + + Fructose + + + + meso -Inositol + + + + Raffinose +---L-Rhamnose + + + + Xylitol + + + + Xylose + + + + Antibiosis against Aspergilus niger +--+ Bacillus subtilis NCIMB 3610 + +-- Micrococcus luteus NCIMB 196 + + + + Saccharomyces cerevisiae CBS 1171 +-+ + Streptomyces murinus ISP 5091 +-+ +

RF: Rectiflexibiles , +: positive,-: negative,

^a PY, pale yellow; G, gray; GW, gray to white; DG, dark gray; O, olive; OP, orchid mixed with pinkish white; VY, violet to yellow; BW,

brown to white; SB, slight browning; W, white.

Showed the Streptomyces strain VC-A46 are other actinomycetes, and other specific properties are relevant G + C content as 75.5% in the strain identified, fatty acid analysis _{C 16: 0 iso, C 16} : 0 and C _{16: 1} cis 9 dominated (see Table 2).

   Fatty acid composition content (Content,%) C _{14: 0} iso 3.81 C _{15: 0} iso 1.01 C _{15: 0} anteiso 4.15 C _{15: 0} 2.83 C _{16: 1} iso H 2.79 C _{16: 0} iso 47.82 C _{16: 1} cis 9 10.24 C _{16: 0} 14.44 C _{17 : 1} anteiso C 0.79 C _{17: 0} iso 0.61 C _{17: 0} anteiso 5.05 C _{17: 1} cis 9 3.36 C _{17: 0} cyclo 0.82 C _{17: 0} 1.55 C _{18: 1} cis 9 0.73

* C _{16: 1} iso: 14-methylpentadecanoic acid

* C _{16: 0} : hexadecanoic acid

* C _{16: 1} cis 9: cis-9-hexadecanoic acid.

Genomic DNA of the strain was extracted according to the method of Pospiech and Neumann, and then amplified 16S rDNA using primers fD1 (AGAGT TTGAT CCTGG CTCAG: SEQ ID NO: 2) and rP2 (ACGGC TACCT TGTTA CGACTT: SEQ ID NO: 3). The sequence was analyzed and the nucleotide sequence (SEQ ID NO: 1) is shown in FIG.

The analyzed sequences were compared with those of other actinomycetes in GenBank, and 16S rDNA of Streptomyces sp.

Through the above physiological and biochemical tests and 16S rDNA sequencing, the actinomycetes strain of the present invention was identified as Streptomyces cheonanensis sp.nov ., And Streptomyces cheon-ansis VC-A46 ( Streptomyces). cheonanensis sp.nov.VC -A46).

As described above, the present invention provides a novel Streptomyces Cheonan Ensis VC-A46 strain having an antimicrobial activity against phytopathogens and a fungicide for controlling plant diseases using the same. The effect which can control environmentally friendly can be aimed at.

Attach sequence list electronic file  

Claims (4)

Streptomyces cheonanensis sp.nov.VC -A46 strain (microbial accession number: KCCM 10641P) having antibacterial activity against phytopathogens. A fungicide for controlling plant diseases, comprising the strain of claim 1 or a culture filtrate of the strain. The method of claim 2, wherein the phytopathy is Phytophthora capsici , Colletotrichum orbiculare , Magnaporthe grisia , Fusarium oxysporum , A plant disease caused by any one of Alternaria mali and Rhizoctonia solani . A plant disease control method for controlling plant diseases by treating a plant with a bactericide comprising the strain according to claim 1 or a culture filtrate of the strain.

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