KR100512480B1 - A novel Streptomyces sp. lim7 having protecting ability for clubroot - Google Patents

Abstract

The present invention relates to a novel Streptomyces sp. Lim7 strain (KACC91025) having a control effect against cabbage wart disease, a method for controlling cabbage wart disease using the strain, and a biochemical pesticide preparation containing the strain.

The strain according to the present invention is useful as a biopesticide because it has an effect of controlling Chinese cabbage wart disease better than the conventional chemical pesticides, without destroying the ecosystem due to overuse that may occur in conventional chemical pesticides.

Description

Novel Actin Streptomyces sp. A novel Streptomyces sp. lim7 having protecting ability for clubroot}

The present invention relates to a novel Streptomyces sp. Lim7 (KACC91025) strain having a control effect against cabbage wart disease, a method for controlling cabbage wart disease using the strain, and a biochemical pesticide preparation containing the strain.

There are 4,157 pests that cause damage to crops grown in Korea, but 1,199 species (bottle 574 and 625 pests) have been confirmed to occur at present. It is bringing reality.

As these pests cause damage to crops, one of the easiest means to exclude or control them from crops is called pesticides, which are recognized as natural when pests and weeds occur on crops. It is. However, the negative view is likely to increase gradually as pesticides, which are convenient control measures, have recently been presented with harmful issues such as safety issues, residual problems (crops, soil, environment), problems on the natural ecosystem, and pollution.

Even though there are various problems, it is impossible to prevent the use of all pesticides currently in use. Has been studied.

The research history of controlling pests using microorganisms was the first attempt to control beetle pests with a fungus in Germany in 1880. Since then, many studies have been carried out and about 70 species have been put to practical use as microbial pesticides worldwide.

Looking at the current state of development in foreign countries, many researches have been attempted as biopesticides using microorganisms themselves. This is a pest control action using microorganisms itself. Representative studies using antagonistic microorganisms are representative.

Bio pesticides were the first to use actinomycetes to control potato beetle disease in the United States in 1927. In 1951, a study was carried out on virus control using cross-protection and sweet potato stem rot using mold.

In addition, in Korea, antimicrobial antibiotics, which are effective against rice blast, gray bacterium cabbage, and Candida, have been isolated (Kim, Kwan, Kim, Chang Hwan, 1989, Acetoxycycloheximide). Biological Activity and Its Production Strains, Journal of the Korean Society for Industrial Microbiology, 17: 307-312).

Cabbage wart was first discovered in Europe in the 13th century, and the pathogen causing it ( Plasmodiophora brasicae Woron.) Was reported by M. Woronin in 1877 and is widely distributed in Europe and Asia. In Korea, there was a record of the first outbreak in Suwon and Seoul in September 1928, but this was not a problem. However, the disease has been increasing every year since 1991 in Goyang, Gimpo, Pyeongtaek, and Jeonbuk Muju in Gyeonggi-do. In 1994, 30% of Goyang in Gyeonggi-do was impossible to harvest. Cabbage wart disease is 6-7 years in the soil, so the infection is spread quickly through the infected soil as well as infected crops. In addition, since it is spread in the form of dormant spores, it is difficult to control. The best control method is crop rotation. Because cabbage warts pathogens grow well in acidic soils, lime treatment is also used as a control method to neutralize soil acidity. Drug control of Chinese cabbage wart disease was used after two drugs were announced in 1996, but only about 70% of the control effect was shown.

Therefore, the present inventors have shown an excellent effect on the control of Chinese cabbage wart disease, and as a result of intensive efforts to develop environmentally friendly biochemical pesticides, it was confirmed that Streptomyce sp. The present invention has been completed.

An object of the present invention is to provide a novel Streptomyces sp. Lim7 (KACC91025) strain having a control effect against cabbage wart disease.

Another object of the present invention to provide a method for controlling cabbage wart disease using the strain.

Still another object of the present invention is to provide a biochemical pesticide preparation using the strain and a preparation method thereof.

In order to achieve the above object, the present invention provides a Streptomyces sp . Lim7 (KACC91025) strain having a control effect against cabbage wart disease.

The present invention also provides a Streptomyces sp . Strain having a control effect against cabbage wart disease and having a 16S rRNA sequence of SEQ ID NO: 1.

The present invention also provides a method for producing a biochemical pesticide preparation, characterized in that the above strain is cultured. The culture may be characterized in that performed for 7 to 14 days at 25 ~ 30 ℃ in Bennett (Bennet) medium.

The present invention also provides a method for controlling cabbage wart disease, characterized in that using the strain.

The present invention also provides a biochemical pesticide preparation comprising the strain or its culture as an active ingredient. The culture solution may be characterized in that it is incubated for 7-14 days at 25 ~ 30 ℃ in Bennett (Bennet) medium, the biochemical pesticides may be characterized in that for the control of cabbage wart disease.

Streptomyces genus of the present invention (Streptomyces sp.) Lim7 strains, the Republic of Korea on the 13th March 2003 Agricultural Biotechnology nine won the strain of (Gwonseon-gu Suwon seodundong 225 (R) 441-707) in actinomycetes (Streptomyces sp.) Lim7 the name Deposited under accession number KACC91025.

The invention is briefly described below.

The novel Streptomyces sp. Lim7 (KACC91025) of the present invention is an antimicrobial bacterium isolated from soil, characterized in that the gene base sequence structure of 16S rRNA has the following specific structure. Has a homology of 98% with S. bikiniensis in the whole nucleotide sequence of and has excellent control effect in cabbage wart disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Novel Actinomycetes Streptomyces sp.) Isolation and Identification of KACC91025

Culture medium

Bennett medium used for actinomycetes culture was used for suspension culture of selected Streptomyces sp. Lim7 (KACC91025). The medium was composed of Agar 15 g / L, Beef extract 1 g / L, Yeast extract 1 g / L, Tryptone 2 g / L, glycerol 10 g / L. Incubated for 10 days.

Morphological Identification

Pure strain isolated Streptomyces sp. Lim7 (KACC91025) strains were cultured in Bennet medium and observed with a scanning electron microscope (Fig. 1).

16S rRNA gene base sequence analysis

Genetic analysis of the novel Streptomyces sp. Lim7 (KACC91025) was performed, and sequencing of the 16S rRNA gene was performed as follows. The cells were separated by suspension culture for 7 days in Bennet's medium for DNA extraction and centrifugation of the culture. DNA extraction was performed using Genomic DNA Extraction Kit (Intron).

PCR was carried out in a reaction solution (10 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl ₂ ; 0.2 mM dNTPs; 1 unit Taq DNA polymerase). Amplified by The amplified fragments were about 1500 base-pairs in size. After PCR reaction (initial 94 ℃ 5 minutes once; 94 ℃ 1 minutes, 58 ℃ 1 minutes, 72 ℃ 2 minutes, 35 cycles, 72 ℃ 10 minutes once) and amplified 16S rRNA gene fragment pGEM-T Easy vector After ligation to (Promega.co.), E. coli DH5α was transformed.

Transformed E. coli was provoked in a selection medium (1.5% agar, 50mg / L ampicillin, 100μM IPTG, 50mg / L X-gal) and incubated for 12 hours at 37 ℃. Insertion of the vector containing the amplified 16S rRNA gene fragment was cultured by inoculating only LB medium with white colony in a medium showing blue / white colony. The culture was centrifuged for plasmid separation and Alkaline lysis was used. The isolated plasmid was digested with restriction enzyme Eco RI and confirmed insertion of amplified 16S rRNA fragment through electrophoresis.

The amplified 16S rRNA gene sequence was analyzed by a sequencing device (ABI3100) after direct sequencing reaction of PCR fragments using four sequencing primers (Table 1) and Big dye terminator v.3.0 (Applied Biosystems). As a result, it has the nucleotide sequence of SEQ ID NO: 1. This homology was confirmed using BLAST of the National Center for Biotechnology Information (NCBI). The results showed homology of 98% with Streptomyces bikiniensis (FIG. 2).

Primers used for PCR and sequencing of 16S rRNA genes primer Primer Sequence PCR fD1 (SEQ ID NO: 2) 5'-AGA GTT TGA TCC TGG CTC AG-3 ' rP2 (SEQ ID NO: 3) 5'-ACG GCT ACC TTG TTA CGA CTT-3 Sequencing p510r (SEQ ID NO: 4) 5'-TAT TAC CGC GGC TGC TG-3 p364f (SEQ ID NO: 5) 5'-GGC AGC AGT GGG GAA TAT TG-3 p783f (SEQ ID NO: 6) 5'-TAG ATA CCC TGG TAG TCC AC-3 p1037f (SEQ ID NO: 7) 5'-TCG TCA GCT CGT GTC GTG AG-3

Example 2 Radiation Strain ( Streptomyces sp.) Effect of lim7 (KACC91025) culture medium on the control of cabbage wart disease

Black cabbage ( Brassica campestris subsp. Napus var. Pekinensis , cv Hukjinju) was used for the test and the cabbage wart bacteria was Plasmodiophora brassicae Woron. Seeds were planted in horticultural tops (side farms), grown in greenhouses, so that two-leafed cabbages were planted, and then cabbage root diseased tissue was placed in a waring blender and sterile water was added and ground. This was filtered with a cheese cloth to prepare dormant spore solution of Plasmodiophora brassicae , and the dormant spore solution was mixed well with the field soil to prepare two bland soils. The concentration of spores was adjusted to 1 × 10 ⁶ concentration per ml of soil, 100 ml of dry soil was put in a plastic pot of 6.5 cm in diameter, and then 2-leafed cabbage was put thereon, and 50 ml of byeongto was added again. The microorganisms cultured on the cabbage transplanted to Lee Byeong soil were poured 10 ml per pot. The untreated group treated only the medium in which the microorganisms were not cultured. Pharmaceutically treated cabbages were grown in a greenhouse (20 ± 5 ° C.) for about 21 days to develop cabbage clubroot.

The disease was investigated with the following onset indices.

 The incidence index is 0 when no lumps occur, the incidence index is 1 when a small nodules occur in the apex, and the incidence index is 2 when a large or small nodules are formed in the apex, or a large nodules in the apex and aides. In this case, the incidence index was investigated.

In addition, the control value was calculated according to the following equation.

                           Average incidence index of treatment group

    Control Value (%) = (1-────────────────) X 100

                          Average Incidence Index of Untreated Zone

The results of treatment of the culture solution (containing microbial intracellular material) of Streptomyces sp . Lim7 (KACC91025), which were cultured by the method of Example 1, were compared with the untreated group (FIG. 3). In general, a cabbage wart disease caused by Plasmodiophora brassicae causes a lump in the root.

Referring to FIG. 3 is perhaps due to the Streptomyces genus (Streptomyces sp.) Lim7 (KACC91025 ) culture treated (left) and untreated (right) both been infected by Plasmodiophora brassicae untreated is a lump was found in the roots treatments antifungal activity of bacteria No occurrence was observed.

The experimental results showed that the cabbage wart control effect was 100% when Streptomyces sp . Lim7 (KACC91025) culture medium was treated. From this, it was confirmed that the KACC91025 bacteria of the present invention has an excellent cabbage wart control effect.

The microbial agent of the present invention can be prepared and used in the form of a variety of preparations including the culture or culture of the strain or strain of the present invention, that is, liquids, hydrates, powders, granules, emulsions and the like.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has an effect of providing a novel Streptomyces sp. Lim7 (KACC91025) bacteria having a cabbage wart disease control effect, a method for controlling cabbage wart disease using the same and a biochemical pesticide preparation containing the strain.

The strain according to the present invention is useful as a biopesticide because it shows an effect of controlling Chinese cabbage wart disease better than the conventional chemical pesticides, without destroying the ecosystem due to overuse that may occur in conventional chemical pesticides.

1 is a scanning electron micrograph of a novel Streptomyces sp. Lim7 (KACC91025) strain.

Figure 2 shows the results of 16S rRNA gene analysis of the novel Streptomyces sp. Lim7 (KACC91025).

Figure 3 is a photograph showing the results of treatment of the culture solution of Streptomyces sp. Lim7 (KACC91025) to the cabbage with cabbage wart disease, the left side is Streptomyces sp. The result of treatment of the culture medium of lim7 (KACC91025) was shown.

<110> KonKuk University <120> A novel Streptomyces sp. lim7 having protecting ability for clubroot <130> KUP03-036 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 1490 <212> DNA <213> Streptomyces sp. lim7 <400> 1 agagtttgat cctggctcag gacgagcgct ggcggcgtgc ttaacacatg caagtcgaac 60 gatgaagccc ttcggggtgg attagtggcg aacgggtgag taacacgtgg gcaatctgcc 120 cttcactctg ggacaagccc tggaaacggg gtctaatacc ggatacgagt ctgggaggca 180 tctcctggac tggaaagctc cggcggtgaa ggatgagccc gcggcctatc agcttgttgg 240 tggggtaatg gcctaccaag gcgacgacgg gtagccggcc tgagagggcg accggccaca 300 ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat attgcacaat 360 gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg ttgtaaacct 420 ctttcagcag ggaagaagcg aaagtgacgg tacctgcaga agaagcgccg gctaactacg 480 tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc cggaattatt gggcgtaaag 540 agctcgtagg cggcttgtca cgtcgggtgt gaaagcccgg ggcttaaccc cgggtctgca 600 tccgatacgg gcaggctaga gtgtggtagg ggagatcgga attcctggtg tagcggtgaa 660 atgcgcagat atcaggagga acaccggtgg cgaaggcgga tctctgggcc attactgacg 720 ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 780 acgttgggaa ctaggtgttg gcgacattcc acgtcgtcgg tgccgcagct aacgcattaa 840 gttccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga cgggggcccg 900 cacaagcagc ggagcatgtg gcttaattcg acgcaacgcg aagaacctta ccaaggcttg 960 acatataccg gaaagcatta gagatagtgc cccccttgtg gtcggtatac aagtggtgca 1020 tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080 tgtcctgtgt tgccagcatg cccttcgggg tgatggggac tcacaggaga ccgccggggt 1140 caactcggag gaaggtgggg acgacgtcaa gtcatcatgc cccttatgtc ttgggctgca 1200 cacgtgctac aatggccggt acaaagagct gcgatgccgt gaggcggagc gaatctcaaa 1260 aagccggtct cagttcggat tggggtctgc aactcgaccc catgaagtcg gagttgctag 1320 taatcgcaga tcagcattgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380 cacgtcacga aagtcggtaa cacccgaagc cggtggccca accccttgtg ggagggagct 1440 gtcgaaggtg ggaccagcga ttgggacgaa gtcgtaacaa ggtagccgta 1490 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 3 acggctacct tgttacgact t 21 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer <400> 4 tattaccgcg gctgctg 17 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer <400> 5 ggcagcagtg gggaatattg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer <400> 6 tagataccct ggtagtccac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer <400> 7 tcgtcagctc gtgtcgtgag 20

Claims (8)

Streptomyces sp . Lim7 (KACC91025) strain having control effect against cabbage wart disease. delete A method for producing a cabbage wart disease control agent, comprising culturing the strain of claim 1. The method of claim 3, wherein the culturing is carried out for 7-14 days at 25-30 ° C in a Bennett medium. Method of controlling cabbage wart disease, characterized in that using the strain of claim 1. Chinese cabbage wart control agent comprising the strain of claim 1 or a culture thereof as an active ingredient. [Claim 7] The cabbage wart control agent according to claim 6, wherein the culture solution is incubated for 7 to 14 days at 25 to 30 DEG C in a Bennett medium. delete