KR100896041B1 - A novel Staphylococcs sp. and biopesticide composition comprising the strain against tomato or potato late blight - Google Patents

Abstract

The present invention relates to a novel strain belonging to the genus Staphylococcus sp. And a composition comprising the strain, and more particularly, to isolate an antimicrobial material against tomato late blight or potato late blight in salted fish The present invention relates to the investigation of antimicrobial substances from a Staphylococcus bacterium and its use as agricultural microorganisms.

Staphylococcus genus, tomato late blight, potato late blight, agricultural microbial preparation

Description

A novel strain belonging to the novel Staphylococcus genus and a composition for controlling tomato or potato late blight comprising the strain {A novel Staphylococcs sp. and biopesticide composition comprising the strain against tomato or potato late blight}

The present invention relates to a novel strain belonging to the novel Staphylococcus sp. ( Staphylococcus sp.) And a composition comprising the strain, and more particularly to tomato blight or potato blight, which has recently been seriously damaged in tomato cultivation complexes. In order to develop an antimicrobial substance against the present invention, the present invention is to investigate the antimicrobial substance from Staphylococcus bacterium isolated from salted fish and to use it as an agricultural microbial agent.

Commonly Phytophthora Tomato and potato late blight caused by infestans are very damaging and occur in low and humid growing areas. P. infestans can invade the ground foliage of potatoes and tomatoes at any time during the growing season, killing ground crops, and also invading potato tubers and tomato fruit and rotting during packaging and storage (Judelson, HS 1997. The genetics and biology of Phytophthora infestans: modern approaches to a historical challenge.Fungal.Genet. Biol. 22: 65-76; Judelson, HS, BM Tyler, and RW Michelmore. 1991. Transformation of the oomycete pathogen, Phytophthora infestans.Mol.Plant.Microbe.Interact 4: 602-607). In addition, the plague is mainly airborne, which almost completely destroys all plants on the package within 1-2 weeks when the weather conditions are low and high. If the disease is not controlled, the economic damage is well documented by the Irish Great Famine, an epidemic of potato plague in Ireland in 1845, in which 1.5 million people died of famine and 2 million moved to the New World. In 1863, by deBary, the causative agent of the disease was P. infestans , an oomycetes. Since its discovery, numerous studies have been conducted to control the disease.

As a control method for controlling tomato late blight, disease-free seed potatoes, resistant varieties, timely drug spraying, etc. have been mainly relied on chemical control by disinfectant spraying. Resistant varieties are used as a supplementary tool in controlling pests, such as reducing the frequency of drug spraying as resistance is easily broken by the emergence of new races.

However, bacteria with resistance to metalaxyl and dimethomorph, which are excellent penetrant fungicides, were generated and their density gradually increased. In order to suppress this resistant bacterium, the overspray of the drug causes environmental problems, and in recent years, the development of environmentally friendly new control agents is required as the interest in the environment increases rapidly. In particular, as consumer demand for eco-friendly agricultural products increases, it is necessary to develop biopesticides, that is, microbial pesticides and biochemical pesticides, which have excellent control effects for eco-friendly agricultural products.

It is an object of the present invention to solve the above problems and to be devised by the necessity of the present invention is to provide a new agricultural microorganisms.

In order to achieve the above object, the present invention provides a bacterium belonging to the genus Staphylococcus sp . Showing a control effect against tomato or potato late blight.

The strain of the present invention is preferably Staphylococcus sp . AP27, Staphylococcus sp . AP27, Staphylococcus bacteria in the Korea Agricultural Biotechnology Park (Dec. 22, Seodun-dong, Gwonseon-gu, Suwon-si, Gyeonggi-do 441-707) Genus ( Staphylococcus sp .) Was deposited under the strain name AP27 and accession number KACC91291P.

In another aspect, the present invention provides a microbial pesticide preparation comprising a strain belonging to Staphylococcus sp . Having a pathogenicity against tomato or potato late blight as an active ingredient.

The strain used in the microbial pesticide formulation of the present invention is preferably Staphylococcus sp . AP27.

The invention is briefly described below.

The present invention firstly provides a novel Staphylococcus sp. AP27 strain,

Second, Staphylococcus Species provides Staphylococcus Species AP27 bacteria, it characterized in that it has a unique structure, such as to the gene arrangement of the 16S rRNA of AP27 bacteria, (Staphylococcus sp.) (Staphylococcus sp.) - total Sequence homology: 98% homology with Staphylococcus pasteuri .

Third, Staphylococcus sp. Staphylococcus sp. Provides a method for preparing a culture medium of AP27 bacteria,

Fourth, there is provided a agricultural microbial agent, characterized in that the treatment of the culture solution for the control of tomato or potato blight.

The culture and biochemical properties of the Staphylococcus spp. Of the present invention are generally the same as the culture and biochemical properties of the Staphylococcus genus known in the art.

The present invention shows that the culture medium of the novel Staphylococcus sp. AP27 can be used as an agricultural microorganism, which is an ecosystem due to overuse that may appear in tomato or potato blight control in conventional chemical pesticides. The effect is to prevent the destruction of.

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

Example 1

[Isolation of Novel Staphylococcus sp. AP27 from Salted Fish]

Salted fish was used as sample. The strains were used for the separation of the ilrusyeon method (dilution method), first the sample Fermented stock solution 10 ^-1 - 10 ^- and smeared by a 100ul diluted with sterilized distilled water to nutrient agar (nutrient agar) to ^three 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 Staphylococcus sp. AP27]

Selected strain AP27 was used for the culture broth (Nutrient broth) medium used for culturing Staphylococcus bacteria 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

[Formal Observation of Novel Staphylococcus sp. AP27]

The purified Staphylococcus sp. AP27 strain isolated from pure culture was cultured in nutrient broth (Nutrient broth) medium and observed with a scanning electron microscope (Fig. 1).

Example 4

[Analysis of 16S rRNA Gene Base Sequence of New Staphylococcus sp. AP27]

Genetic analysis of the novel Staphylococcus sp. AP27 bacterium 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). The results showed 99% homology with Staphylococcus pumilus .

 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

[Effect of Staphylococcus sp. AP27 Bacteria on the Control of Tomato Blight]

Test subject tomato ( Lycopersicon Breed esculentum Mill.) were used for tomatoes purchased from Aurora Co. heungnong seedling tomato pathogen is Phytophthora Station infestans (Mont.) de Bary. Tomato seeds were planted in horticultural soil (vice farms), grown in greenhouses, and used for experiments after cultivation to tomato seedlings of two to three leaves. Pathogen Phytophthora infestans PIT strains were inoculated in oatmeal agar and incubated for 7 days in a dark state at 20 ° C. incubator for 7 days while irradiating with light for 16 hours a day to form sporangia. I was.

By spraying the microorganism culture solution of AP27 strain on Seedling tomato processing of a medicament and air drying of that after, the addition of sterilized distilled water to the culture medium and harvesting the yujujanang and 3x10 ⁴ per ml to examine the blood cells to step spore concentration under a light microscope one yujujanang for 1 day The suspension was made at low temperature after the suspension was discharged, and then sprayed onto the treated tomato seedlings. Tomato seedlings inoculated with the pathogen were incubated for 1 day in a 20 ° C. chamber, and then placed in a constant temperature and humidity chamber (70% relative humidity) for 2 days. After examining the area ratio of the lesions formed in each tomato seedling, the control value was calculated according to the following equation.

[Equation]

Control value (%) = {(Legn area ratio of 1-treated area / Long-area area of untreated area) × 100}

The results of treatment of the culture medium (containing microorganisms of intracellular material) of Staphylococcus sp. AP27 cultured by the method described in Example 2 were compared with the untreated (FIG. 3). In general, when a tomato late blight is caused by Phytophthora infestans , the necrosis of the tomato leaves is wilted. Referring to FIG. 3, in the non-treated group (left), tomato late blight caused by the inoculated Phytophthora infestans , and the tomato leaves withered, and the cultured treatment group of AP27 (right) had a bactericidal effect on the Phytophthora infestans of AP27 culture. Tomato leaves are healthy with no wilting symptoms.

As a result, when the culture medium of Staphylococcus sp. AP27 was treated, tomato pest control effect was 95%. As a control drug, 100ppm of chlorothalonil was used, which showed 100% control value.

As a result of the above, the strain and the composition have Phytophthora having the same origin as tomato late blight. It can be seen that it is also effective in potato plague caused by infestans .

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 Staphylococcus sp.) The AP27 strain was cultured in Bennet's medium and observed by Scanning Electron Microscope.

Figure 2 is a novel Staphylococcus sp.) 16S rRNA gene analysis of AP27.

Figure 3 is a staphylococcus genus ( Patphylococcus) in tomato diseased tomato sp.) A photo showing the results of treatment of the culture medium of AP27. Staphylococcus on tomato with tomato late blight sp.) The result of treatment with the culture medium of AP27 (right) and the result without treatment (left).

<110> Konkuk University Industrial Cooperation Corp. <120> A novel Staphylococcus sp. and biopesticide composition          comprising the strain against tomato or potato late blight <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 <400> 6 tcgtcagctc gtgtcgtgag 20  

Claims (7)

delete delete A strain of Staphylococcus sp . AP27 (KACC 91291P), which exhibits excellent control against tomato blight or potato blight. Staphylococcus sp . Staphylococcus sp . AP27 (KACC 91291P) A composition exhibiting excellent antagonistic ability against tomato late blight, characterized in that it contains as an active ingredient. Staphylococcus sp . Staphylococcus sp . AP27 (KACC 91291P) A composition exhibiting excellent antagonistic ability against potato late blight, characterized by containing as an active ingredient. delete delete

Images