KR20090035368A - A novel bacillus sp. and biopesticide composition comprising the strain against clubroot - Google Patents

Abstract

A bio-pesticide composition comprising Bacillus sp. is provided to prevent Chinese cabbage clubroot, thereby restraining ecosystem from being devastated caused by overuse of existing chemical pesticides. A bio-pesticide composition comprises Bacillus sp. separated from fermented seafood. The Bacillus sp. represents Bacillus sp. BA35(KACC 91172P). A gene base sequence structure of 16S rRNA of the Bacillus sp. BA35(KACC 91172P) has 99% homogeny with Bacillus pumilus. The bio-pesticide composition having excellent effects of preventing Chinese cabbage clubroot contains Bacillus sp. BA35(KACC 91172P).

Description

A novel Bacillus sp.and biopesticide composition comprising the strain against clubroot} including a new strain belonging to the genus Bacillus （Ｂａｃｉｌｌｕ ｓ ｓｐ．） and its strain

The present invention is a novel Bacillus genus ( Bacillus The present invention relates to a novel strain belonging to sp.) and a composition comprising the strain, and more particularly, Bacillus bacteria isolated from salted fish to develop an antimicrobial substance against cabbage root-knot disease, which has been seriously damaged in recent cabbage planting complexes. It is an invention for the use as agricultural microorganisms by examining the antimicrobial substance from.

In general, cabbage root-knot disease, which occurs widely in Europe and Asia and causes great damage, is caused by the pathogen Plasmodiophora. Brassicae is a disease caused by cruciferous crops such as cabbage, cabbage, and turnip. The disease was first discovered in Europe in the 13th century, and the pathogen causing the disease was first reported by M. Woronin in 1877. In Korea, there was a record of the first outbreak in Suwon and Seoul in September 1928. However, since 1991, the disease has been increasing in Goyang, Gimpo, Pyeongtaek, and Jeonbuk Muju in Gyeonggi-do, and it is increasing every year. In 1994, 30% of Goyang in Gyeonggi-do was impossible to harvest.

Chinese cabbage root-knot bacteria form a myriad of dormant spores in diseased tissues, which can live for more than a decade if the environment is right in the soil. Therefore, this disease is a soil disease that is difficult to control compared to other plant diseases. In order to control the cabbage root disease, seedling control such as removing or burning sick plants, raising soil acidity with limestone or alkaline solution, strengthening drainage, crops and crop rotation other than cruciferous plants are used.

However, only disease-resistant varieties have been successful, and others have not worked well. The control of Chinese cabbage root gall disease by disinfectant treatment until now three kinds of drugs have been used, but shows only about 70% of the control effect is increasing the demand for a new control agent having excellent control effect.

In addition, as agricultural consumers have increased interest in environmental and human toxicity, the demand for organic agricultural products grown with eco-friendly pesticides such as microbial pesticides and biochemical pesticides is increasing.

SUMMARY OF THE INVENTION The object of the present invention is to solve the above problems and to provide a novel agricultural microbial agent.

In order to achieve the above object, the present invention is a genus of Bacillus ( Bacillus) showing a control effect against the Chinese cabbage root gall sp .) is provided.

The strain of the present invention is Bacillus genus ( Bacillus sp.) BA35 and should preferably, August 27, 2005 Republic of Korea Agricultural Biotechnology nine won genus Bacillus bacteria (Gwonseon-gu Suwon seodundong 225 (R) 441-707) (Bacillus sp .) was deposited under the strain name BA35 and accession number KACC 91172P.

In addition, the present invention has a genus Bacillus ( Bacillus) having pathogenicity against cabbage root gall disease It provides a microbial pesticide formulation, characterized in that it contains a strain belonging to sp .) as an active ingredient.

The strain used in the microbial pesticide preparation of the present invention is Bacillus genus ( Bacillus sp .) is preferably BA35.

The culture, biochemical properties and the like of the strains of the present invention are the same as the culture, biochemical properties of the general genus Bacillus well known in the art.

The invention is briefly described below.

First, the novel Bacillus fungus ( Bacillus sp.) provides BA35 bacteria,

Second, Bacillus sp.) Bacillus genus ( Bacillus) characterized in that the genome sequence structure of the 16S rRNA of BA35 bacterium has the following unique structure: sp.) to provide BA35,-homology of the entire base sequence: 99% homology with Bacillus pumilus.

Third, Bacillus sp.) providing a method for preparing a culture medium of BA35 bacteria,

Fourthly, there is provided an agricultural microbial agent, characterized in that the treatment of the culture solution for the control of Chinese cabbage root-knot disease.

The present invention shows that a novel Bacillus sp. BA35 culture medium can be used as a microbial agent for the control of Chinese cabbage root nodule disease, which is to prevent the destruction of the ecosystem due to overuse that may occur in conventional chemical pesticides. It can be prevented.

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

Example 1

[Isolation of New Bacillus sp. BA35 from Salted Fish]

Salted fish was used as a sample. The strains were used for the separation of the ilrusyeon method (dilution method), the first sample Fermented stock solution 10 ^l - and smeared by 100ul by up to 10 ^-3 dilution with sterile distilled water to nutrient agar (nutrient agar) medium plate, 28 ℃ 3-7 days incubation in the colony was confirmed. The identified colonies were passaged on fresh plate nutrient agar medium. For identification of the isolated strains, optical microscopy was first performed based on Burgy's manual.

Example 2

[Selection of Selection Medium for Cultivation of New Bacillus sp. BA35]

The selected strain BA35 was used as a nutrient broth medium used for culturing Bacillus for suspension culture. The medium is composed of beep extract (Beef extract) 3 g / L, Peptone (Peptone) 5 g / L, the incubation temperature is 28 ℃, pH 6.8 and incubated for 10 days.

Example 3

[New Bacillus sp.) Morphological observation of BA35 bacteria]

A separate pure New Species of Bacillus (Bacillus sp.) The BA35 strain was cultured in nutrient broth (Nutrient broth) medium and observed with a scanning electron microscope (Fig. 1).

Example 4

[New Bacillus sp.) Analysis of 16S rRNA gene base sequence of BA35 bacteria]

Bacillus sp.) Genetic analysis of BA35 bacteria was performed, and sequencing of the 16S rDNA gene was performed as follows. Suspension culture in LB medium for 2 days for DNA extraction and the cells were separated by centrifugation. DNA extraction was performed using Genomic DNA Extraction Kit (Intron).

PCR was performed with 30 ng genomic DNA, 0.25 uM 16S rDNA 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 rDNA 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. Identification of the vector containing the amplified 16S rDNA gene fragment was first selected by selecting only the white colony of the blue / white colony, it was inoculated in LB medium and cultured. The culture was centrifuged for plasmid separation and Alkaline lysis was used.

The isolated plasmid was digested with restriction enzyme E coRI and confirmed the insertion of amplified 16S rDNA fragment by electrophoresis. The amplified 16S rDNA gene sequence was subjected to direct sequencing reaction of PCR fragments using four sequencing primers (Table 1) and Big dye terminator v.3.0 (Applied Biosystems) prepared by our sequencing assay (ABI3100). ) And homology was confirmed using BLAST of the National Center for Biotechnology Information (NCBI) (FIG. 2). As a result Bacillus pumilus ) and 99% homology.

Table 1 shows the 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 5

[Effects of Bacillus sp. BA35 Culture on the Control of Chinese Cabbage Root Gland Disease]

Chinese cabbage campestris subsp. Varieties of pekinensis) were using the dog days of Chinese cabbage are staggered purchased from Ltd heungnong seed was cabbage knot germs Plasmodiophora brassicae Woronin. Seeds are planted in horticultural soils (cultivated) and grown in greenhouses so that two-leafed cabbages are cabbage. After harvesting from cabbage, cabbage root diseased tissue stored in -80 ℃ deep freezer is placed in a waring blender and sterilized. Added and triturated. The tissue was removed by filtration with cheese cloth, and the dormant spores of Plasmodiophora brassicae were prepared. At this time, the concentration of spores was adjusted to 1x10 ⁶ concentration per ml soil. 100 ml of healthy soil was put in a plastic pot having a diameter of 6.5 cm, and two-leafed Chinese cabbage was put on it, and 50 ml of byeongchi was added again. The microorganisms cultured on the cabbage transplanted to Lee Byeong soil were irrigated 30 ml per pot, and the untreated group was treated with the same amount as the treated group without the culture medium. Drug-treated cabbage was grown in a greenhouse (20 ± 5 ° C.) for about 21 days to develop cabbage rootroot disease (clubroot).

After examining the disease with the following incidence index, the control price was calculated according to the following equation. 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.

[Equation]

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

The results of treatment of the culture solution (containing microbial intracellular material) of Bacillus sp. BA35 cultured by the method described in Example 2 were compared with the untreated (FIG. 3). Usually Plasmodiophora When cabbage root gall is caused by brassicae , it forms gall on cabbage root. Referring to Figure 3, in the untreated (right) Plasmodiophora present in the two soils The cabbage was infected by brassicae and nodules were formed in the roots. The culture treatment of BA35 bacteria (left) showed no nodules in the cabbage roots due to the control effect of the cultures.

As a result, Bacillus sp.) The treatment of BA35 bacteria showed 100% cabbage control effect. 167 ppm of fluazinam was used as a control agent, which showed 89% control value.

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.

1 is a novel genus Bacillus sp.) The BA35 strain was cultured in Bennet's medium and observed by Scanning Electron Microscope.

2 is a novel Bacillus genus ( Bacillus sp.) 16S rRNA gene analysis of BA35 bacteria.

Figure 3 is a genus of Bacillus ( Bacillus) cabbage root cabbage root disease sp.) A photograph showing the results of treatment of the culture solution of BA35 bacteria. Bacillus sp.) The results of treatment with BA35 culture medium (right) and untreated (left).

<110> Konkuk University Industrial Cooperation Corp. <120> A novel Bacillus sp. and biopesticide composition comprising the          strain against clubroot <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> fD1 Primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rP2 Primer <400> 2 acggctacct tgttacgact t 21 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> p510r Primer <400> 3 tattaccgcg gctgctg 17 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p364f Primer <400> 4 ggcagcagtg gggaatattg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p783f Primer <400> 5 tagataccct ggtagtccac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p1037f Primer <400> 6 tcgtcagctc gtgtcgtgag 20  

Claims (4)

A microbial strain belonging to the genus Bacillus sp. That exhibits excellent control against Chinese cabbage root-knot disease. According to claim 1, wherein the strain is Bacillus genus ( Bacillus sp .) microbial strain, characterized in that BA35 (KACC 91172P) Bacillus sp .) The composition exhibiting excellent control against the Chinese cabbage root-knot disease, characterized in that it contains a strain belonging to the active ingredient. According to claim 3, wherein the strain is Bacillus genus ( Bacillus sp .) BA35 (KACC 91172P).

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