KR101852462B1 - Streptomyces sp. AN120537 strain having insect juvenile hormone antagonist and insecticidal activities and uses thereof - Google Patents

Abstract

The present invention discloses a Streptomyces sp. AN120537 strain capable of antagonizing a larval hormone by controlling a Met (methoprene-tolerant) receptor or a composition for controlling pests of the strain and uses thereof. The strain or the culture extract according to the present invention is an insect growth regulator derived from actinomycetes, which is an eco-friendly insecticide having an insecticidal effect by specifically acting on the insect and harmless to a plant; and has the same action mechanism and action point as an insect hormone so that the resistance regarding the regulator is difficult to be obtained by the insect.

Description

Streptomyces sp. AN120537 strain having a larval hormone antagonist and insecticidal activity and use thereof {Streptomyces sp. AN120537 strain having insect juvenile hormone antagonist and insecticidal activities and uses thereof,

The present invention relates to Streptomyces sp. AN120537 strains having larval hormone antagonist and insecticidal activity and uses thereof.

Pests are not only economically damaging crops but also affecting humankind by mediating a variety of pathogens that cause disease in humans. The demand for environmentally friendly pesticides is increasing because of the environmental pollution through residues and the toxicity and the resistance to insect pests.

Insect growth regulators (IGRs) are emerging as an alternative to chemical pesticides because they act specifically on target pests and are less toxic to the environment. The insect growth regulator has formed a market of about 6,500 billion won worldwide, and classified into three kinds of larval hormone analogue, exocrine hormone agonist and chitin synthesis inhibitor depending on the action mechanism. Since larval hormones play an important role in regulating insect development, reproduction and various other physiological functions, larval hormone-based insect growth regulators, including larval hormone agonists and antagonists, By disturbing the system, abnormal development and lethality can be induced in the larval stage, which can be effectively used for insect control.

Recently, researches for the development of new environmentally friendly pesticides using plants and microorganisms have been actively carried out all over the world. In particular, actinomycetes produce secondary metabolites such as antibiotics during growth, and secondary metabolites derived from actinomycetes inhibit larval growth and have insecticidal activity. Examples include Spodoptera litura , a secondary metabolite derived from actinomycetes that inhibits normal growth of larvae and exhibits insecticidal activity against the target pest (Talwinder Kaur et al., Insecticidal and growth inhibitory potential of Streptomyces hydrogenans DH16 on major pest of India, Spodoptera litura (Fab.) (Lepidoptera: Noctuidae). BMC Microbiology . 14: 227. 2014). However, strains of actinomycetes having larval hormone antagonist activity and insecticidal activity thereof have not yet been reported.

Korean Patent Application No. 10-2014-0100066 discloses a method for screening antagonists of insect hormones and a composition for an insecticide, which comprises the step of culturing insect larval hormone receptor Methoprene-tolerant (Met) and transformant transformed with CYC or SRC And a composition for insecticide using the same.

Korean Patent No. 10-1720472 relates to a composition and a method for controlling insect pests through the regulation of Met receptor including a plant-derived larval hormone agonist or antagonist, and it relates to an antagonist of an agonist of a larval hormone Or an antagonist, and a method for controlling the insecticide using a new insect growth regulator derived from a plant.

Korean Patent No. 10-1101783 relates to a novel strain of Streptomyces sp. Having excellent antimicrobial activity and insecticidal activity, a method of culturing the same, and a method of using the culture and a culture product. The present invention relates to a novel antimicrobial activity and insecticidal activity against plant pathogens and insects And a method for producing a preparation using the strain.

However, as insect pests that show resistance to currently commercialized insect growth regulators are beginning to appear, it is necessary to develop new and effective insect growth regulators.

The present invention relates to a strain of Streptomyces sp. AN120537 having insecticidal activity simultaneously against larval hormone antagonist activity and Aedes albopictus and Plutella xylostella larva, and a method of culturing the above strain and a pest control using a culture extract or And an object of the present invention is to provide a control method.

In one embodiment, the present invention relates to Streptomyces sp. Strain AN120537 deposited on Dec. 12, 2017 with KCTC 13420BP at the Life Resource Center, Korea Research Institute of Bioscience and Biotechnology.

In another embodiment there is provided a larval hormone antagonist composition comprising a Streptomyces strain AN120537 strain according to the present invention or an extract of a culture of said strain.

In another aspect, there is provided a composition for modulating Met receptor comprising Streptomyces sp. Strain AN120537 according to the present invention or an extract of a culture of the strain.

In another embodiment there is provided a larval hormone antagonist composition comprising a strain of Streptomyces sp. AN120537 according to the present invention or an extract of a culture of said strain.

The strain according to the present invention can be used as an extract of a cultured organism or a cultured organism.

In one embodiment, the extract of the culture is not limited to, but is not limited to, hexane or ethyl acetate, especially hexane.

In another aspect, there is provided a composition for controlling pests, through antagonistic activity against a larval hormone comprising a Streptomyces strain AN120537 strain according to the present invention or an extract of a culture of said strain.

Pests that are effective in the composition according to the present invention are those that express the larval hormone and the Met (Methoprene-tolerant) receptor protein, including, but not limited to, lepidoptera or dicotyledonous, boll weevil, It is not.

In another embodiment, the present invention provides a method of treating or preventing a Streptomyces spp. Strain AN120537 or a culture extract thereof according to the present invention from insect pests, insect larvae or insect pests; All or part of a plant, or seed of a plant; Or contacting a pest or plant habitat.

In another embodiment, the present invention provides a method of treating Metoprene-tolerant Met (Met), a larval hormone receptor in the pests, comprising treating Streptomyces spp. AN120537 or an extract of a culture of the strain with insect pests, larvae or insect pests, Lt; RTI ID = 0.0 > receptor. ≪ / RTI >

The strains of Streptomyces sp. AN120537 according to the present invention and their cultured extracts have insect larval hormone antagonistic activity and are resistant to conventional insecticides by using the larval hormone antagonistic activity having an insecticidal mechanism different from those of the existing IGR series materials Can be effectively used to control pests.

In addition, the culture filtrate of this strain does not have the larval hormone antagonist activity, whereas the cell extract only has the larval hormone antagonist activity. Thus, the present strain has a great advantage in recovering larval hormone antagonist activity and insecticidal activity, because the larval hormone antagonist activity and the insecticidal activity substance are not secreted into the culture filtrate but exist in the cells.

The extract of actinomycetes cultured according to the present invention is an eco-friendly insecticide which has an insecticidal effect on a target insect but is completely harmless to the plant itself and acts on the same action mechanism and action point as an insect hormone. It is advantageous in that it can be effectively used for the control of pests resistant to existing pesticides.

FIG. 1 shows the results of evaluation of the larval hormone antagonistic activity of the three extracts obtained by culturing the strain of Streptomyces sp. AN120537 according to the present invention in a solvent fraction.
FIG. 2 shows the results of evaluation of the antihistokinetic activity of the three extracts obtained by culturing the strain of Streptomyces sp. AN120537 according to the present invention in a solvent fraction.
FIG. 3 shows the results of insecticidal activity against three third instar larvae of three extracts obtained from a culture of Streptomyces sp. Strain AN120537 according to the present invention through a solvent fraction.
FIG. 4 shows the results of insecticidal activity against the third instar larva of Cabbage moths of three extracts obtained from the culture of Streptomyces sp. Strain AN120537 according to the present invention through a solvent fraction.

The present invention is based on the discovery of a novel strain of Streptomyces sp. AN120537 with antagonist and insecticidal activity of insect larval hormone.

Thus, in one embodiment, the present invention relates to Streptomyces sp. Strain AN120537 deposited on Dec. 12, 2017 with KCTC 13420BP at the Life Resource Center, Korea Research Institute of Bioscience and Biotechnology.

Streptomyces sp. Strain AN120537 according to the present invention was isolated from forest soil as a native microorganism in Korea. As a result of molecular systematic analysis based on 16s rRNA gene sequence, Streptomyces celluloflavus NRRL B-2493 (T) and 99.03% similarity (EZ-taxon blasting results). The strain isolated above was designated as Streptomyces sp. AN120537 strain and deposited with KCTC 13420BP on December 12, 2017 at the Korea Research Institute of Bioscience & Biotechnology.

The strain according to the present invention has antagonistic activity against insect larval hormone.

The Juvenile Hormone (JH) of the present invention regulates the transformation process that periodically takes off the exoskeleton during the development of insects. In the case of the adult, the female is normally involved in the production of eggs and acts through the receptor The receptor is a protein that exists in the body of an insect, detects and recognizes the larval hormone, and performs the function so that the hormone can play a role.

The antagonist of the present invention binds to the larval hormone receptor and inhibits the action of the substance. It inhibits the activity of the insect hormone to induce abnormal insect metamorphosis or inhibits normal egg differentiation, . The insect larval hormone antagonist according to the present invention can be used as an insecticide for insect pest control by inhibiting normal molting of larvae or by inhibiting normal ovarian development of adult insects.

Strain according to the present invention Streptomyces spp. AN120537 strain can be used as a culture medium itself or an extract of the above culture medium.

The method of culturing the strain for the production of the culture of the strain according to the present invention and the preparation of the extract can be referred to those described in the examples herein.

In one embodiment, the culture itself may be used. In this case, however, the cell membrane or the like may be broken in the environment in which the cultured material is treated, and the substance in the cell may leak out of the cell and produce an effect.

In other embodiments, an extract of the culture may be used. The culture extract may be used in one embodiment as an extract of an organic solvent, especially hexane or ethyl acetate, especially hexane.

In another embodiment, the extract of the culture does not include the extract of the culture filtrate. The cultured filtrate is not effective but is effective only in the cell extract, and the cultured filtrate is not included in the culture because the larval hormone antagonist activity and insecticidal activity are not secreted in the culture filtrate but exist in the cells.

The strain of Streptomyces sp. AN120537 or its culture extract according to the present invention is not limited to this theory, but it can inhibit the abnormal development of the larva by inhibiting the dimer formation between Met and its partner CYC, or inhibit adult reproduction Pests can be controlled. It also affects the hatching rate of insect eggs, and it affects the development of hatching larvae when treated with eggs, which can be effectively used for control.

In one embodiment, the Streptomyces strain AN120537 strain or a culture extract thereof according to the present invention modulates Met as a receptor for JH. Met is a receptor for larval hormones (Jindra, M., Palli, SR & Riddiford, LM The juvenile hormone signaling pathway in insect development. Annu Rev. Entolol 58, 181-204, Ftz- F1-interacting steroid receptor coactivator (FISC) or cycle (CYC) (Li, M., Mead, EA and Zhu, J. Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression. proc. Natl. Acad. Sci. USA 108, 638-643, 2011).

The cultivar extract insecticide of Streptomyces spp. AN120537 according to the present application can be applied to the pest itself, its larvae or eggs which need to be controlled, or to all or a part of the area, habitat or plant where control is required.

The strain according to the present invention has an antagonistic activity to the larval hormone by acting on Met, a receptor of the larval hormone.

However, larval hormones of insects are not different depending on the insect species, but most of them have almost the same chemical structure, and the receptor protein Met of the larval hormone has similar characteristics. Therefore, the larval hormone antagonistic activity of the strain cultures according to the present invention is generally effective for insects having larval hormone and Met receptor protein.

A pest in which a strain according to the present application or a composition comprising it is used refers to an invertebrate or microorganism that damages a plant or animal through colonization, damage induction, or infection. Pests are also individuals that compete with host and nutrients, damaging or spreading disease.

In another embodiment according to the present application, in particular pests associated with plants, it also comprises fungi, bacteria, insects, arachnids, nematodes, slugs, snails and the like or eggs or larvae of these insects. Other embodiments according to the present invention also include crops, natural environments and / or domestic pests and eggs and larvae thereof.

In one embodiment according to the present application, the pest includes hygiene pests and agricultural pests that express the larval hormone and Met receptor protein.

In another embodiment according to the present application, various insects such as aphids, beetles, and thrips, which are mainly problematic in domestic as well as lepidoptera and paralysis, all have larval hormone and Met receptor protein. For example, Egyptian forest mosquitoes ( Aedes aeypti ), white mosquito ( Aedes albopictus ), red house mosquitoes ( Culex pipiens ), small root flies ( Bradysia agrestis , Spodoptera exigua , Plutella xylostella , Spodoptera litura ), the American white moth ( Hyphantria cunea , Helicoverpa armigera , Plodia interpunctella , Chilo suppressalis , Cnaphalocrocis medinalis , Ostrinia furnacalis , Riptortus clavatus , Lissorhoptrus, oryzophilus ), brownfly ( Nilaparvata lugens), aemyeolgu (Laodelphax striatellus), cotton aphid (Aphis gossypii), Green Peach Aphid (Myzus persicae ), cucumber thrips ( Thrips palmi ), flower yellow thrips ( Frankliniella occidentalis), or tobacco Louis (Bemisia tabaci), such as one including arthropods, or Al or larvae of the insects which the larvae hormone present, not intended to limit this, larvae which can serve the composition according to the present hormone receptor and It can be applied to all insects with a helper.

Plants to which the insecticide according to the present application may be applied may be applied to whole plants or parts thereof, for example to the cells or tissues of plants as well as roots, stems, leaves, and seeds. Crops to which pesticides according to the present invention can be applied include crops (wheat, barley, rye, oats, rice, sugar cane, etc.), beets (sugar beet and feed beet), grass (orchard grass, Lentils, peas, beans), oil plants (oilseed rape, mustard, poppy, olive, sunflower, coconut, castor, apple, pear, plum, peach, almond, cherry, strawberry, raspberry, blackberry) Vegetable plants (cotton, flax, hemp, jute), citrus fruits (tangerine, orange, lemon, grapefruit, etc.), vegetables (spinach, (Eg, lettuce, asparagus, cabbage, onion, tomato, potato, paprika, etc.), camphor (avocado, carrot, cinnamon, camphor), hardwoods and conifers (eg linden-tree, , Birch, fir, larch, pine), or corn, tobacco, nuts, coffee, candy Number, include vegetation or ornamental plants such as tea, vines, hops, bananas, and natural rubber. Also, the plant or crop to which the composition according to the invention is applied comprises both planted or growing crops and harvested crops.

Control, control or control of insect pests by an insecticide herein includes inhibiting or interfering with the eradication or pest control of the target pest physically or its growth or behavior. Thus, it includes eradicating pests, preventing the production of sperm or eggs through the generation of insectless genera, or damaging insect genes, thereby rendering the sperm or egg unfit to adult.

The composition for pest control or insecticide of the present invention contains the active ingredient of the extract of the strain and / or the culture of the strain, but may further include excipients and additives contained in other conventional compositions for insecticides. For example, it may include, but is not limited to, a plasticizer, a colorant, a brightener, a surfactant, a dispersant, an emulsifier, a fluidizing agent and the like.

The composition for insecticide of the present invention can be formulated into a conventional insecticide or pesticide formulation. For example, the composition for insecticide of the present invention can be formulated into an emulsion, a liquid agent, a wetting agent, a water-soluble agent, a powder, a granule, a fumigant, a coating agent and the like. Specifically, the compounds of formulas (1) to (8) are formulated into pesticide formulations by titration. These composition formulations are described in "Catalog of pesticide formulation types and international coding system" 2, 6th Ed. May 2008, CropLife International]. The composition is described in Mollet and Grubemann, Formulation technology, Wiley VCH, Weinheim, 2001; Or in the known manner as described in the Knowles, New developments in crop protection product formulation, Agrow Reports DS243, T & F Informa, London, 2005.

Specifically, a surfactant, an extender, a disintegrant, a nutrient, etc. may be mixed and pulverized into an extract of a culture of a strain and / or a strain to be used as a wetting agent or dissolved to form a liquid formulation. Surfactants include, but are not limited to, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkylphenyl Ether, sodium tripolyphosphate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene aryl phosphoric ester, But are not limited to, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyethylene alkylaryl polymer specials, polyoxyalkylone alkyl phenyl ethers, Ethylene nonyl phenyl ether (polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, Triton 100, and Tween 80 may be used. Examples of surfactants are listed in McCutcheon's, Vol. 1: Emulsifiers & Detergents, McCutcheon's Directories, Glen Rock, USA, 2008 (International Ed. Or North American Ed.). As the disintegrant, bentonite, talc, kaolin, calcium carbonate and the like can be used. As the extender, one or more of loess, diatomaceous earth, dextrin, glucose and starch can be used.

The composition for pest control or insecticide of the present invention may contain an insecticidally effective amount of a compound, for example, the amount of the pesticidal compound to be used may be 0.01 to 100 (mg / kg). The term "effective amount" means an amount of a composition or compound that is sufficient to control larvae on a cultivated plant or is sufficient for the protection of the material and does not cause substantial damage to the treated plant. Such amounts can vary within wide limits and depend on a variety of factors, such as the pest species to be controlled, the plant or material being treated, the climatic conditions and the particular compound used. When the formulation is used, it can be used at any time when a pest is generated.

In another aspect, the present application is also directed to a method of treating Metoprene-tolerant (or Metoprene-tolerant) Met of the insect or insect larva, comprising treating the insect or pest larva with a culture extract of Streptomyces sp. Strain AN120537, Lt; RTI ID = 0.0 > receptor. ≪ / RTI >

In another embodiment, the present application is also directed to a method of treating a pest or insect larvae Met (Methoprene-Pyridoxine) of the pest or insect larva, comprising treating Streptomyces sp. Strain AN120537 or a culture extract thereof or a composition comprising the same, tolerant < / RTI > receptor.

In the method according to the present invention, the modulation of the larval hormone is by antagonism.

In the method according to the present application, we can also refer to the above-mentioned control of larval hormone by regulating Met (Methoprene-tolerant) receptors of Streptomyces sp. Strain AN120537 or its culture extract according to the present invention.

In another embodiment, the present application provides a composition for insecticides comprising the Streptomyces sp. Strain AN120537 and / or a cultured extract thereof, in combination with a pest or a habitat of the pest, or a plant, soil or water containing plant Lt; RTI ID = 0.0 > habitat. ≪ / RTI >

The pests and crops to which the method may be applied are as described above.

The pest control or control of the present invention refers to the removal of pests or the removal or removal of pests by preventing or relieving the crops from pests.

The method of the present invention can be used to control pests or their larvae or their eggs themselves, or habitats of pests; Or to a habitat in which the whole or part of the plant or its seed, plant or insect or its larvae live, for example, soil or water, by any application method known in the art . In this case, "contact" means direct contact (compound / composition applied to pests or plants - typically directly to plant leaves, stems or roots) and indirect contact (compound / composition applied to pests or plant nursery ) ≪ / RTI >

The methods herein may also be used to protect a plant / crop from attack or intrusion by insect pests by contacting the growing plant or crop with an insecticidally effective amount of the composition. As used herein, the term plant or crop includes both the growing crop and the harvested crop.

The methods herein are utilized by treating an insecticidally effective amount of a plant, plant propagation material, such as seed, soil, surface, material, or space, to be protected from attack by a pest, with the present composition. The methods herein can be carried out both before and after infection of plant, plant propagation material such as seed, soil, surface, material or space by insect pests.

The method can be prophylactically applied where pest incidence is expected.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental material

The Y187 cell transformed with Met and CYC used in the present invention ( Saccharomyces cerevisiae strain Y187-Aa; accession number: KCTC12621BP) was the one disclosed in Korean Patent Laid-Open No. 10-2014-0100066.

Example  1. Isolation of actinomycetes according to the present invention from soil

The actinomycetes were cultivated in the soil of Humic acid - vitamin agar (0.1% humic acid: dissolved in 0.2N NaOH, 0.05% Na ₂ HPO ₄ , 0.171% KCl, 0.005% MgSO ₄ , 0.001% FeSO ₄ 8 vitamins and 2% agar including 7H ₂ O, 0.002% CaCO ₃ , 50 ppm cycloheximide, 10 ppm nalidixic acid, thymine-HCl) (Hayakawa, M. and Nonomura, H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes, J. Ferment, Tech., 65: 501-509, 1987). For the preservation of isolated actinomycetes, cultures were grown in Bennett's medium (1% glucose, 0.1% yeast extract, 0.2% Bacto-peptone, 0.1% beef extract) (Hesseltine, CW, Benedict, RG and Pridham, TG Useful criteria for species differentiation in the genus Streptomyces . Ann. NY Acad . Sci . 60: 136-151, 1954).

Example  2. Identification of isolated strains

The AN120537 strain isolated in Example 1 was analyzed for genetic and cultural characteristics through 16S rRNA sequences as follows.

(1) Cultural characteristics

In order to analyze the culture characteristics according to various media of the strain, the culture physiological characteristics of the strain were examined after culturing at various temperatures of ISP medium for 14 days at 28 ° C. As a result, the growth of strains of Streptomyces sp. AN120537 showed relatively good growth in yeast - malt agar, oat mill agar and peptone - yeast - iron agar. Inorganic salts - starch agar, glycerol - asparagine agar and tyrosine agar It was normal. In the peptone - yeast - fermentation agar, a water - soluble pigment of pinkish purple color was produced, and a pale yellow pigment was produced in the agar of tyrosine.

[Table 1] Cultural physiological characteristics of Streptomyces sp. Strain AN120537 by culture medium

(2) Genetic characteristics

Sequence analysis of 16s rRNA gene was performed to isolate isolates. Specifically, genomic DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen) to obtain the nucleotide sequence of the 16s rRNA gene of the strain. 27F (5'-AGAGTTTGATCMTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3 ') primer. The amplified PCR product was purified and subjected to a sequence analysis by BIOFACT Corp. The nucleotide sequence was compared with the nucleotide sequence in the GenBank database using the EzTaxon-e server (http://www.ezbiocloud.net) and the NCBI BLAST. As a result, strain AN120537 showed 99.03% similarity with Streptomyces celluloflavus NRRL B-2493 (T). The isolated strain was named Streptomyces sp. AN120537, and deposited on December 12, 2017 with the Life Resource Center of the Korea Research Institute of Bioscience and received the deposit number KCTC 13420BP.

Example  3. According to the present invention Streptomyces sp. ( Streptomyces sp .) AN120537 Lactic Hormone Antagonist Activity Assay of Cultured Strains

The insect larva hormone antagonist activity of Streptomyces sp. AN120537 according to the present invention was tested using the system disclosed in Korean Patent Application No. 2014-0100066 filed by the present inventor. When the transformant transformed with Met and CYC was cultured in the presence of the extract of the strain cultured according to the present invention, it was determined that Met and CYC did not bind and thus the antagonistic activity of the larval hormone was observed when LacZ was not transcribed .

The concrete experimental method is as follows. Genus Streptomyces (Streptomyces (2% soluble starch, 1% glucose, 1% soybean meal, 0.02% FeSO ₄ .7H ₂ O, 0.3%) were added to the strains of the strain AN120537 for the assay of the larval hormone antagonist activity according to the solvent fraction of the strain AN120537. % CaCO ₃ (pH 7.2)) at 28 ° C and 150 rpm for 7 days. After culturing, the culture was centrifuged to collect the culture, and the same amount of acetone was added to the culture, followed by standing for 24 hours. The mixture was centrifuged for 24 hours, and the supernatant was centrifuged at Whatman No. 1. The filtrate was filtered under reduced pressure. The filtrate was concentrated under reduced pressure at 50 ° C (EYELA, Japan), followed by solvent fractionation using an equal volume of water, hexane, ethyl acetate and butanol. Hexane, ethyl acetate and butanol extracts were obtained from the solvent fraction and concentrated under reduced pressure. Each extract was dissolved in dimethylsulfoxide to investigate the antagonistic activity of the larval hormone.

For assay of the larval hormone antagonist activity, yeast Y187 cells transformed with Met-CYC of Egyptian mosquitoes (S accharomyces S. cerevisiae strain Y187-Aa; Accession No: KCTC12621BP; Korean Patent Laid-Open No. 10-2014-0100066) was cultured at 30 ° C in a DDO (SD-Leu / -Trp) medium until the OD ₆₀₀ became 0.3-0.4. In this way the return of the cultured cells 2.0 × 10 ⁶ cells / ml as the concentration of suspended in fresh medium, and distribute it in a 96-well plate by 100㎕. Three extracts of 0.033ppm of pyrimethoxyphene and one strain of Streptomyces sp. AN120537 were cultured in a 96-well plate at a concentration of 10ppm and further cultured at 30 占 폚 for 3 hours. Thereafter, an East? -Galactosidase assay kit (Thermo Scientific) was used according to the manufacturer's method to perform? -Galactosidase analysis. As a positive control group, 0.033 ppm of pyrimethoxyphene and 10 ppm of kanakugiol were treated, and 0.033 ppm of pyrimethoxyphenol and 10 ppm of dimethylsulfoxide and pyrimethoxyphene were treated as negative control groups, and β-galactose The syndadic analysis was performed. These β-galactosidase assay reaction mixtures in 96-well plates were incubated at 30 ° C. for 5 hours, and each OD ₄₂₀ was measured using an iMark ^™ microplate reader (BIO-RAD). The thus measured OD ₄₂₀ was substituted into the following formula 1 to evaluate the larval hormone antagonist (JHAN) activity of each extract. The results are shown in the horizontal axis of FIG.

[Equation 1]

JHAN activity = (OD ₄₂₀ control-OD ₄₂₀ AN120537) / OD ₄₂₀ control

In the above formula 1, the OD ₄₂₀ control was 0.033 ppm of pyrimethoxifene and OD ₄₂₀ when the solvent dimethylsulfoxide was treated. OD ₄₂₀ AN120537 contained 0.033 ppm of pyrimethoxyphene and 3 extracts of Streptomyces sp. AN120537, of the OD ₄₂₀ in a case where processing in density.

The above equation 1 is an evaluation of the OD ₄₂₀ of three extracts when 0.033 ppm of pyrimethoxifene and OD ₄₂₀ of the case of treatment with dimethylsulfoxide solvent were regarded as zero.

As a result, as shown in FIG. 1, when the extract of the strain of Streptomyces sp. AN120537 according to the present invention was assayed for the larval hormone antagonist activity by controlling the Met receptor for three extracts, the hexane extract was found to be about 0.8 level at a concentration of 10 ppm High larval hormone antagonist activity. The ethyl acetate extract showed about 0.3 larval hormone antagonist activity at the concentration of 10 ppm, while the butanol extract showed no larval hormone antagonist activity.

Example  4. Harvest (anti-yeast) active absence verification

This experiment was conducted to demonstrate that the larval hormone antagonist activity of the strain according to the present invention in Example 3 is not caused by anti-yeast activity but by the strain according to the present invention.

When the substance to be investigated is toxic to yeast, the OD ₄₂₀ determined by the method of Example 1 is not due to the larval hormone antagonistic activity of the substance of interest, but is due to anti-yeast activity. Three yeast cultures of Streptomyces sp. AN120537 strain of Example 1 were tested for anti-yeast activity by examining growth inhibitory effects on yeast cells.

For this purpose, yeast Y187 cells transformed with Met-CYC in Egyptian mosquito were cultured at 30 ° C. in a DDO (SD-Leu / -Trp) medium until the OD ₆₀₀ reached 0.3-0.4. In this way the return of the cultured cells 2.0 × 10 ⁶ cells / ml as the concentration of suspended in fresh medium, and distribute it in a 96-well plate by 200㎕. 0.03 ppm and 10 ppm of pyriproxyphene, 10 ppm of cancousiol and 3 ppm of extracts were treated at a concentration of 10 ppm and shake incubation was performed at 30 ° C for 3 hours to determine the growth rate of the cells for one day on a 96-well plate ₆₀₀ .

As a result, as shown in Fig. 2, the genus Streptomyces sp.) AN120537 cultures did not have any effect on the growth of yeast cells, indicating no anti-yeast activity.

Example  5. Hygiene pest  Insecticidal Activity Test for Dicotyledonous Pest

The insecticidal activities of mosquito larvae were evaluated for three extracts of strains of Streptomyces sp. Strain AN120537 in Examples 1 and 2, and the results are shown in Fig. Aedes albopictus) third instar larvae of the port 10 rats treated, treated with a concentration of 10 ppm for each of the three extracts to investigate the number of mortality for 3 days every 24 hours. As a control, larval hormone analogue (pyriproxyphen) and larval hormone antagonist (cancusiol) were used, and treated at a concentration of 10 ppm.

As a result, the genus Streptomyces of the present invention (Streptomyces sp.) AN120537 showed a high level of insecticidal activity against the third instar mosquito larvae, and 100% insecticidal activity even at a concentration of 10 ppm.

Example  6. Agricultural pest  Lent Insecticidal activity against pests  black

The insecticidal activity against the larvae of the Chinese cabbage larvae, which are a pest insect, was evaluated using three extracts of Streptomyces sp. Strain AN120537. The results are shown in Fig.

Three extracts were treated with 10 ppm of each of the three moth larvae of the cabbage moth, and the mushroom water was collected for 24 hours at 3 - day intervals. As a result, it was confirmed that the hexane extract had an excellent insecticidal activity at 20 ppm, showing 100% insecticidal activity against the third instar larva (Fig. 4). As a control, larval hormone analogue (pyriproxyphen) and larval hormone antagonist (cancusiol) were used and similarly treated at a concentration of 20 ppm for 3 days at 24 hour intervals.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (13)

Streptomyces sp. Strain AN120537 deposited with KCTC 13420BP at the Life Resource Center of the Korea Research Institute of Bioscience and Biotechnology.
11. A larval hormone antagonist composition comprising the Streptomyces strain AN120537 strain according to claim 1 or an extract of a culture of said strain.
3. The method of claim 2,
Wherein the extract of said cultures does not include culture filtrate.
3. The method of claim 2,
Wherein the extract of said culture is an extract extracted with a hexane solvent.
A composition for controlling insect pest or diarrhea through antagonistic activity against a larval hormone comprising the Streptomyces sp. Strain AN120537 according to claim 1 or an extract of a culture of said strain.
delete 6. The method of claim 5,
The parachute pests are Aedes aeypti, Aedes albopictus, Culex pipiens, or Bradysia agrestis,
The above-mentioned lepidopteran pests include Spodoptera exigua, Plutella xylostella, Spodoptera litura, Hyphantria cunea, Helicoverpa armigera, Plodia interpunctella, , Chilo suppressalis, Cnaphalocrocis medinalis, or Ostrinia furnacalis.
A strain of Streptomyces sp. AN120537 according to claim 1, or a culture extract thereof, is applied to a pest of a lepidoptera or dicotyledonous species; All or part of a plant, or seed of a plant; Or contacting said pest or plant habitat.
9. The method of claim 8,
Wherein the extract of the cultured product does not contain culture filtrate.
9. The method of claim 8,
Wherein the extract of said cultured product is an extract extracted with a hexane solvent.
delete 9. The method of claim 8,
The parachute pests are Aedes aeypti, Aedes albopictus, Culex pipiens, or Bradysia agrestis,
The above-mentioned lepidopteran pests include Spodoptera exigua, Plutella xylostella, Spodoptera litura, Hyphantria cunea, Helicoverpa armigera, Plodia interpunctella, , Chilo suppressalis, Cnaphalocrocis medinalis, or Ostrinia furnacalis.
delete

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