KR20050028699A - A novel streptomyces sp. and biopesticide comprising the strain - Google Patents

Abstract

A microorganism Streptomyces sp. and biopesticide comprising the same microorganism are provided, which microorganism Streptomyces sp. has improved antimicrobial activity against Plasmodiophora brasicae Woron., so that destruction of an ecosystem by improper use of conventional agricultural chemicals can be inhibited. The microorganism Streptomyces sp. (KACC 91020) having improved antimicrobial activity against various microorganisms, especially Plasmodiophora brasicae Woron. is provided, wherein Streptomyces sp. (KACC 91020) is isolated from the soil. The biopesticide comprises Streptomyces sp. (KACC 91020) as an effective component.

Description

A novel microorganism belonging to the novel actinomycetes, and an agricultural microbial agent comprising the strain thereof A A St Streptomyces sp. and Biopesticide comprising the strain}

The present invention relates to a novel microorganism belonging to the novel Streptomyces sp. And agricultural microorganisms comprising the strain, and more particularly, to antibacterial activity against cabbage wart disease, which has been seriously damaged in recent cabbage cultivation complexes. In order to develop a substance, the present invention is to investigate the antimicrobial substance from actinomycetes isolated from soil and to use it as an agricultural microorganism.

Cabbage wart disease 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 has a 6-7 year survival time in soil, so it spreads rapidly through 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. The drug control of cabbage wart disease was used after the two drugs were announced in 1996, but only about 70% of the control effect was shown.

Therefore, it is necessary to develop a new control method.

An object of the present invention is to provide a new agricultural microbial agent to solve the above problems.

In order to achieve the above object, the present invention provides a bacterium belonging to the Streptomyces sp . Showing pathogenicity against cabbage wart disease.

Strain of the present invention in Streptomyces (Streptomyces sp.) In Actinomycetes (Streptomyces sp.) And preferably in the KACC91020, January 10 il Republic of Korea Agricultural Biotechnology saved (Gwonseon-gu Suwon seodundong 225 (R) 441-707) 2003 lim 2 was deposited under the strain name and accession number KACC91020.

In another aspect, the present invention provides a microbial pesticide preparation comprising a strain belonging to the Streptomyces sp . Having pathogenicity against cabbage wart disease as an active ingredient.

The strain used in the microbial pesticide formulation of the present invention is preferably Streptomyces sp . KACC91020.

The invention is briefly described below.

The present invention provides a novel Streptomyces sp. KACC91020 strain, and second, the gene base sequence structure of the 16S rRNA of Streptomyces sp. KACC91020 strain has the following specific structure: Actinomycetes ( Streptomyces sp.) KACC91020 to provide,-Homologous homologous sequence: homology of S. bikiniensis ( S. bikiniensis) with 98% homology. Third, there is provided a method for preparing a culture solution of Streptomyces sp. KACC91020, and fourth, an agricultural microbial agent characterized by the treatment of the culture solution for the control of cabbage wart disease.

Hereinafter, the present invention will be described in detail through non-limiting examples.

Example 1

[Selection of Selection Medium for Cultivation of Novel Streptomyces sp. KACC91020]

The selected strain KACC91020 used Bennet's medium used for actinomycetes for suspension culture. 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. Incubate for 10 days.

Example 2

[Formal Observation of Novel Streptomyces sp. KACC91020]

The pure Streptomyces sp. KACC91020 strain isolated from the cultures in Bennet medium was observed by scanning electron microscopy (Scanning Electron Microscope) (Fig. 1).

Example 3

[A 16S rRNA Gene Sequence Analysis of Novel Streptomyces sp. KACC91020]

Genetic analysis of Streptomyces sp. KACC91020 strain was performed, and sequencing of the 16S rRNA gene was performed as follows. The suspension was cultured in Bennet medium for 7 days for DNA extraction, and then the cells were separated by centrifugation. DNA extraction was performed using Genomic DNA Extraction Kit (Intron). PCR was performed using 30 ng genomic DNA, 0.25 uM 16S rRNA gene primer (fD1 and rP2) in 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 using. 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), E. coli DH5α was transformed. Transformed E. coli was provoked in a selection medium (1.5% agar, 50mg / L ampicillin, 100uM IPTG, 50mg / L X-gal) and incubated at 37 ℃ for 12 hours. 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 EcoRI and confirmed insertion of amplified 16S rRNA fragment through electrophoresis (FIG. 2). The amplified 16S rRNA gene sequence was sequenced by direct sequencing reaction of PCR fragments using four sequencing primers (Table 1) and Big dye terminator v.3.0 (Applied Biosystems). ), And homology was confirmed using BLAST of the National Center for Biotechnology Information (NCBI). The results showed 98% homology with Streptomyces bikiniensis .

Table 1 Primers used for PCR and sequencing of 16S rRNA genes

Primer Primer sequence PCR fD1 (SEQ ID NO: 1) 5'-AGA GTT TGA TCC TGG CTC AG rP2 (SEQ ID NO: 2) 5'-ACG GCT ACC TTG TTA CGA CTT Sequencing p510r (SEQ ID NO 3) 5'-TAT TAC CGC GGC TGC TG p364f (SEQ ID NO: 4) 5'-GGC AGC AGT GGG GAA TAT TG p783f (SEQ ID NO: 5) 5'-TAG ATA CCC TGG TAG TCC AC p1037f (SEQ ID NO: 6) 5'-TCG TCA GCT CGT GTC GTG AG

Example 4

[Effects of Controlling Cabbage Wart on the Culture of Streptomyces sp. KACC91020]

Black cabbage ( Brassica campestris subsp. Napus var. Pekinensis , cv Hukjinju) was used for testing and the cabbage wart 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. Drug-treated cabbage was grown in a greenhouse (20 ± 5 ° C.) for about 21 days to develop cabbage wart disease (clubroot). The disease was investigated with the following incidence index and the control price was calculated according to Equation 1. 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 of 3 was investigated. The results of treatment of the culture solution of Streptomyces sp . KACC91020 cultured with the method described in Example 1 (containing microbial intracellular material) were compared with the untreated (FIG. 3). In general, a cabbage wart disease caused by Plasmodiophora brassicae causes a lump in the root. Looking at Figure 3, both culture treatment (left) and untreated (right) of the culture medium of KACC91020 bacteria were infected by Plasmodiophora brassicae, so no lumps were observed in the roots and no lumps occurred due to the control effect.

[Equation 1]

Control value (%) = {(average incidence index in 1-treated group / average incidence index in untreated group) x 100}

As a result, when the culture solution of Streptomyces sp . KACC91020 was treated, the cabbage wart disease control effect was 100%.

The microbial agent of the present invention can be prepared in the form of various preparations, including liquids, hydrates, powders, granules, emulsions, and the like, including the cultures or cultures of the strains or strains of the invention. The manufacturing method is according to the general manufacturing method in the art.

The present invention shows that the culture medium of novel Streptomyces sp. KACC91020 can be used as a microbial agent for the control of cabbage wart disease, which can prevent the destruction of the ecosystem due to the overuse that may occur in conventional chemical pesticides. It can be prevented.

1 is a result of observing the novel Streptomyces sp. KACC91020 strain cultured in Bennet medium with a scanning electron microscope (Scanning Electron Microscope).

Figure 2 shows the results of 16S rRNA gene analysis of novel Streptomyces sp. KACC91020.

Figure 3 is a photograph showing the results of treatment of the culture solution of actinomycetes (Streptomyces sp.) KACC91020 to the Chinese cabbage with cabbage wart disease. The results of treatment with the culture solution of Streptomyces sp. KACC91020 on cabbage with cabbage wart disease (left) and untreated (right).

<110> KONKUK UNIVERSITY <120> A novel Streptomyces sp. and Biopesticide comprising the strain <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR Primer (fD1) <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR Primer (rP2) <400> 2 acggctacct tgttacgact t 21 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer (p510r) <400> 3 tattaccgcg gctgctg 17 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> Sequencing primer (p364f) <400> 4 ggcagcagtg gggaatattg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> Sequencing primer (p783f) <400> 5 tagataccct ggtagtccac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer (p1037f) <400> 6 tcgtcagctc gtgtcgtgag 20

Claims (4)

A strain belonging to the Streptomyces sp . Pathogenic bacterium for cabbage wart disease. The method of claim 1, The above strain is Streptomyces sp . Strains characterized in that KACC91020 A microbial pesticide preparation, comprising as an active ingredient a strain belonging to the Streptomyces sp . Showing pathogenicity against cabbage wart disease. The method of claim 3, wherein The above strain is Streptomyces sp . Microbial pesticides, characterized in that KACC91020.