KR101402215B1 - Composition for controlling weeds in crops including Streptomyces scopuliridis KR-001, its culture broth, its extract, its fraction as an active ingredient - Google Patents

Abstract

The invention of the novel strain Streptomyces Scott Lee pulley disk (Streptomyces The present invention relates to a strain of KR-001 and a use thereof, and more particularly to a strain of Streptomyces scopolridis KR-001 of the present invention, a culture thereof, an extract of the culture, a fraction of the culture or extract This herb can be used as a useful weed control agent by exerting an excellent fungicide on weeds, weeds, weeds and weeds.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for controlling weeds, which comprises Streptomyces scopululidis strain KR-001, a culture thereof, an extract of the above culture, a fraction of the above culture or an extract, its culture broth, its extract, its fraction as an active ingredient}

The present invention relates to a novel strain of Streptomyces scopuliridis and herbicidal use of the strain.

Weeds or weeds are plants that are not absolute and are not appropriate for the time and place that has arisen as human beings began their agricultural life in relative terms. Because weeds are many kinds and have various weeds, they are divided into weeds, weeds, weeds, weeds and weeds according to plant taxonomic classification and morphological classification. Weeds are divided into annual weeds, annual weeds and perennial weeds. Therefore, in general, weeds are classified into weeds, perennial weeds, perennial weeds, perennial weeds, perennial weeds, and perennial weeds and perennial weeds and perennial weeds, and on the contrary, they are classified into weeds, weeds, weeds, weeds or perennial weeds, weeds, .

Grasses There are wells in the stem of the weeds, between the node and the node, the leaves are separated from the node, and the leaves are divided into leaf sheath and leaf blade which surrounds the stem and protects it. Leaves are narrow and long, with leaf vein in parallel. Blood, barnacles, tussock mackerel, lanceolate, reed, and aphids. The weeds belonging to the sedge are similar to those of the carpenter, but most of them are triangular in shape and have no leaflets or leaf ears. Leaves are narrow, ridgeline, pointed end, small number of small flowers. You are a thriller, you are a thriller, a gangbang, a lance, and a tadpole gangrue. Broad leaves Weeds are plants that do not belong to weeds, weeds, weeds and weeds. Leaves are mainly elliptical, ovate, lanceolate, and the veins are intertwined like net. This category includes many weeds such as mangoes, shamrocks, wormwood, cucumbers, radishes, water strangeness, phlegm,

Sciyos angulatus L. is a 1-year-old naturalized plant of Pak and its origin in North America. It is a weed that seriously destroys the ecosystem as it occurs collectively on the riverside, the roadside, the roadside, The ecosystem disturbance is designated as a wild plant in the Enforcement Rule of the Plant and Material Protection Act. Because it grows with a lot of seed production and grows as a community, it inhibits photosynthesis by covering the herbaceous plants as well as the trees and destroys the soil moisture and nitrogen, disturbing the existing vegetation and changing the biodiversity In addition, the thorns on the epidermis of the seeds directly cause injury to the human being, such as causing skin diseases. In fact, the current spike is distributed all over the country, and distribution area spreads gradually, causing serious damage, and development of control technology for control thereof is urgently required.

To date, the method of removing visible leaves is to cut or plant young plants, which requires a great deal of labor. The use of non-selective organic synthetic herbicides such as paraquat or glyphosate in some croplands may allow some control, but the main source and area of the rhizosphere may be riverside, roadside or people active The control using the actual organic synthetic herbicide is inevitably limited in reality.

Furthermore, due to the emergence of resistant weeds and the potential impacts on ecosystems and environmental pollution as a result of the continuous use of organic synthetic herbicides, and as interest in environmentally friendly agriculture has increased as income and quality of life have been emphasized Since regulations are being tightened around the world in order to reduce the use of synthetic pesticides, it is becoming more difficult to control the visible poultry, which occurs mainly in living areas, using conventional organic synthetic herbicides.

Accordingly, there is a desperate need for a technology for environmentally friendly control of visible spots by using natural or biochemical agents having low toxicity on phosphorus axis and easy decomposition under natural conditions.

As an environmentally friendly herbicide, soil microorganisms such as Streptomyces spp. Or active materials derived from plant-derived natural products are used.

Examples of foreign studies on natural herbicides derived from soil microorganisms and plants include the use of Colletorichum gloeosporioides , Phytophthora Raise (Pseudomonas palmivora), Pseudomonas geulradi gladioli ) as a herbicidal herbicide for Zygomycetes japonica, Ivy and ivy, and for the control of herbicides. In the case of research on herbicidal active substances in plants, anthraquinone, naphtha, Recent studies have reported that natural compounds based on naphthoquinone or benzoquinone inhibit hydroxyphenylpyruvate dioxygenase (HPPD).

Although efforts to discover herbicidal active materials derived from natural materials have been continuously carried out, most microbial herbicides or plant-derived herbicides currently under research and development are predominantly on weeds occurring in agricultural lands.

Accordingly, the present inventors have made efforts to find an environmentally friendly natural herbicide herbicide, and have found that a novel Streptomyces scopolulidis scopuliridis KR-001 strain and found that the strain, its culture, the culture extract, the culture broth or the fraction of the extract and the fraction exhibit excellent fungicidal activity on weeds, weeds, light weeds and heating weeds, The present invention has been accomplished by confirming that it can be effectively used as an effective ingredient of a weed control agent.

It is an object of the present invention to provide a novel strain of Streptomyces scopuliridis .

It is still another object of the present invention to provide a herbicidal herbicidal composition containing at least one selected from the group consisting of the above strain, a culture solution thereof, an extract of the culture solution, a culture solution or an extract fraction thereof and an active fraction thereof And a method for controlling weeds using the composition.

In order to achieve the above object, the present invention relates to a method for producing Streptomyces < (R) > scopuliridis KR-001 strain.

In addition, the present invention relates to a method for producing a recombinant vector, which comprises selecting a strain selected from the group consisting of Streptomyces scopoliliidis KR-001 strain deposited with the deposit number KCTC 12156BP, a culture thereof, an extract of the culture, a fraction of the culture or extract, There is provided a herbicidal composition for controlling weeds, which contains at least one of them as an active ingredient.

In addition, the present invention relates to a method for producing a mutant strain, which is selected from the group consisting of Streptomyces scopoliliidis KR-001 strain deposited with Accession No. KCTC 12156BP, a culture thereof, an extract of the culture, a fraction of the culture or extract, and an active fraction of the fraction Or more to a weed, its seed or its habitat.

The culture of Streptomyces scopuliridis KR-001 deposited with the deposit number KCTC 12156BP of the present invention, the culture thereof, the culture extract, the culture medium or the fraction of the extract or the active fraction of the above fraction can be used as a herb, Light weedy grasses, weed weeds, weed control weeds, and so on, and thus can be usefully used as herbicidal compositions for weed control and weed control.

Figure 1 is a graph showing the activity of Streptomyces scopulirides (deposited with Deposit No. KCTC 12156BPStreptomyces scopuliridis) This figure shows the phylogenetic tree by 16S rRNA gene sequence of KR-001 strain.
Fig. 2 is a graph showing the herbicidal activity of the strain culture broth of the present invention on the basis of concentration.
Fig. 3 is a graph showing the herbicidal activity of each fraction (hexane, ethyl acetate, butanol and water fraction) of the strain culture broth of the present invention on the foliar treatment at the concentration of foliage:
Hexane Fr .: hexane fraction;
EtOAc Fr: ethyl acetate fraction;
BuOH Fr. butanol fraction;
AQ Fr: water fraction;
CK: negative control.
FIG. 4 is a graph showing the herbicidal effect of the ethyl acetate fraction of the culture broth of the present invention upon soil treatment.
FIG. 5 is a graph showing the herbicidal activity of the ethyl acetate fraction of the culture broth of the present invention by concentration at the time of foliar treatment. FIG.
Fig. 6 is a graph showing the activity of the viscous oil in the foliar treatment of the ethyl acetate fraction of the strain of the present inventionSciyos angulatus) By concentration.
FIG. 7 is a graph showing the effect of the viscous effect of the ethyl acetate fraction of the strain according to the present invention on the greenhouse condition through in vivo transfer.
Fig. 8 is a graph showing the effect of controlling the visibility of the ethyl acetate fraction of the strain of the present invention under the packaging conditions during the foliar treatment. Fig.
FIG. 9 is a graph showing the distributionHumulus japonicus Sieb. &Amp; Zucc.) Of the strain of the present invention in terms of concentration, time after fraction treatment, and the like.
Fig. 10 is a graph showing the distributionArtemisia princes Pampan) of the strain of the present invention in the foliar treatment of the ethyl acetate fraction according to concentration and time after fraction treatment.
Fig. 11 is a graph showing the relationship betweenEquisetum arvense L.) showing the effect of controlling the foliar treatment of the ethyl acetate fraction of the strain of the present invention by concentration and time after fraction treatment.
Fig. 12 is a graph showing the relationship between the cloverTrifolium repens L.) showing the effect of controlling the foliar treatment of the ethyl acetate fraction of the strain of the present invention by concentration and time after fraction treatment.
Fig. 13 is a chart showing the weaknesses of the ethyl acetate fraction of the strain of the present invention to the concentration of three kinds of crops such as rice, wheat, barley, and two kinds of light crops such as pepper and tomato in the greenhouse condition.
FIG. 14 is a graph comparing the concentrations of folic acid by concentration of foliar treatment of the ethyl acetate fraction (EA fr.), Bialaphos and glufosinate of the strain of the present invention.
15 is an HPLC chart showing the ethyl acetate fraction of the strain of the present invention.
FIG. 16 is a graph showing the penetration of active agent fractions (EA-1, EA-3, EA-4, EA-5, EA-6 and EA-7) of ethyl acetate of the strain of the present invention Acetate fraction).
Fig. 17 is a graph showing foliar treatment of foliar treatment of EA-3, EA-4 and EA-5 as the result of HPLC analysis of the ethyl acetate fraction of the strain of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to the use of Streptomyces < RTI ID = 0.0 > ( Streptomyces) < / RTI > deposited with Accession No. KCTC 12156BP scopuliridis KR-001 strain.

Streptomyces scopuliridis strain KR-001 (hereinafter referred to as KR-001 strain) according to the present invention was isolated and cultured from forest soil around Daecheong Lake, Cheongwon-gun, Chungcheongbuk-do from Korea Biotechnology Research Institute (KRIBB) And stored at -70 ° C in a deep freezer for freeze-drying.

KR-001 strain in the 16S rRNA gene homology search, Streptomyces Scoring pulley Leadis RB72T with Bar doeeotneun identified as a novel strain showing a homology of about 99.7%, which re-discharge (Streptomyces Mai access Scoring pulley Streptomyces scopuliridis ).

The inventors have Streptomyces Scoring pulley the novel strain Leadis (Streptomyces scopuliridis KR-001, deposited with KCTC on March 09, 2012 and granted the deposit number KCTC 12156BP.

Further, the present invention provides a method for producing a recombinant strain, which comprises selecting Streptomyces scopoliliidis KR-001 strain deposited with the deposit number KCTC 12156BP, a culture thereof, an extract of the culture solution, a fraction of the culture or extract and an active fraction of the fraction Herbicidal herbicidal composition containing at least one of the herbicidal compositions as an active ingredient.

The extract of the culture broth may be obtained through extraction with an organic solvent, preferably C ₁ ~ C ₄ From alcohols (e.g., methanol, ethanol, propanol, butanol, etc.), hexane and ethyl acetate The selected organic solvent may be used alone, or two or more kinds of organic solvents may be sequentially used for extraction.

Preferably, the culture or the fraction of the extract is fractionated successively using hexane, ethyl acetate, butanol, and water as a solvent, but is not limited thereto.

The active fraction is preferably a fraction eluted at 6 to 7 minutes, 8 to 10 minutes, or 15 to 20 minutes using methanol, ethanol or an aqueous solution thereof as a solvent in a column chromatography of the culture broth or fraction of the extract But is not limited thereto.

Preferably, the weeds are selected from the group consisting of weeds, weeds, light weeds, and weed weeds, but are not limited thereto.

The poplar and weed are from Digotaris sanguinalis ), sorghum ( Sorghum bicolor), couch grass (Agropyron smithii) and dolpi (Echinochloa crus - galli ), but the present invention is not limited thereto.

The light leaf weeds were inoculated with Solanum nigrum), Aeschynomene indica (Aeschynomeme indica , Abutilon avicennae , Xanthium strumarium ) and meadows ( Calystegia japonica ), but is not limited thereto.

The heating weeds were weeded with difficulty to control ( Sicyos angulatus , Humulus japonicus ), wormwood ( Artemisia princes , equisetum arvense ) and clover ( Trifolium repens ), but the present invention is not limited thereto.

Wherein the culture broth, its extract, the culture broth or the fraction of the extract or the active fraction of the fraction is selected from the group consisting of Herbicidin A, Herbicidin B and Herbicidin F, Any one or two or more is preferable, but is not limited thereto.

The herbicidal composition for controlling weeds may comprise a carrier and / or a diluent. As used herein, the term " carrier " is intended to encompass an inert, organic, or inorganic material having an active ingredient that is mixed or made with the aim of facilitating its application to, or the storage, transportation and / Material. Examples of such diluents or carriers include, but are not limited to, water, milk, ethanol, mineral oil, glycerol.

The present inventors confirmed that the culture medium of KR-001 strain was treated by foliar application of three kinds of herbaceous weeds and five kinds of light leaf weeds according to the concentration (see Table 3 and FIG. 2).

The present inventors divided the culture into hexane, ethyl acetate, butanol, and water, fractionated by solvent, and treated with four kinds of herbaceous weeds and five kinds of light leaf weeds by foliar application. In the ethyl acetate fraction and butanol fraction, (See Table 4 and Fig. 3).

The present inventors confirmed the viability of the ethylacetate fractions by soil treatment and foliar treatment of the ethyl acetate fraction with four kinds of herbs and weeds and five kinds of light leaf weeds according to the concentrations (see Tables 5 and 6, Figs. 4 and 5) .

The inventors have confirmed that the ethyl acetate fraction was treated leaf to the visible night conditions in the greenhouse hayeoteum completely control the visibility at night and 2000 μg mL ^-1 and 1000 μg mL ^-1 concentration (see Fig. 6).

The inventors of the present invention confirmed that the ethyl acetate fraction had an effect of controlling the viscoelasticity through in vivo transfer under the greenhouse condition (see FIG. 7).

The present inventors confirmed that the ethylacetate fractions were foliarized on the basis of the concentration of the viscous fungi under the packaging conditions and showed excellent germicidal power and that the germicidal powers were improved over time and were excellent in drug efficacy persistence 8).

The present inventors confirmed that the ethyl acetate fraction was subjected to foliar application to the weeds of weevil, horseradish weevil, wormwood, beetle and clover, under the packaging conditions (see Table 8, FIGS. 9, 10, 11 and 12).

The present inventors confirmed that the ethyl acetate fraction was subjected to foliar application to foliage and crops (rice, wheat, barley) and photoperiod (pepper, tomato) 13).

The present inventors have found that, when foliar treatment of the ethyl acetate fraction, Bialapos and Glufosinate at the same concentration, the ethyl acetate fraction is comparable to that of vialaphos and glufosinate (See Fig. 14).

The present inventors detected the active substance of the ethyl acetate fraction by column chromatography using methanol, ethanol or an aqueous solution thereof as a solvent (see FIG. 15). Herbicidine A, Herbicidine B, and Herbicidine F were isolated from the fraction thus detected. In addition, it was confirmed that the detected fractions, that is, the active fractions were excellent in the caulking force (see FIGS. 16 and 17).

Therefore, the Streptomyces scopoliidis KR-001 strain deposited with the deposit number KCTC 12156BP of the present invention, the culture thereof, the culture solution extract, the culture medium or the fraction of the extract and the active fraction of the fractions can be used as a herb, Weeds and heating herbicides, it can be usefully used as an effective ingredient of herbicidal compositions for controlling weeds.

In addition, the present invention relates to a method for producing a recombinant vector, which comprises selecting a strain selected from the group consisting of Streptomyces scopoliliidis KR-001 strain deposited with the deposit number KCTC 12156BP, a culture thereof, an extract of the culture, a fraction of the culture or extract, Treating the weeds, their seeds or their habitats with at least one of the weeds.

The inventors of the present invention can apply the culture liquid, the solvent fraction, and the ethyl acetate fraction to the plants by foliar treatment or soil treatment. Such culture, solvent fractions, and ethyl acetate fractions can be used in the form of powders, coarse dust, micro granules, granules, wettable powders, emulsifiable concentrates, liquid solvents, , Water-degradable granules or oil suspensions. The culture, solvent fractions, and ethyl acetate fractions were found to have excellent fungicidal activity against foliar treatments applied to the stem and leaves of the plants, and soil treatments applied to the soil, such as weeds, weeds, weeds and heating weeds.

Therefore, the Streptomyces scopoliliidis KR-001 strain deposited with the deposit number KCTC 12156BP of the present invention, the culture thereof, the culture solution extract, the culture medium or the fraction of the extract or the active fraction of the fraction can be subjected to foliar treatment or soil treatment It is useful as a weed control method for treating weeds, seeds or their habitats.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention in detail, and the present invention is not limited to the examples.

< Example  1> Isolation of strain

Humic acid-vitamin medium was used for isolation from forest soil around Daecheong Lake, Chungwon-do, Chungcheongbuk-do, and cyclohexamide or nalidixic acid was added at an appropriate concentration to inhibit the growth of bacteria and fungi. Respectively. Each 1 g of soil sample was added to sterilized physiological saline solution (10 ml), mixed thoroughly with vortex, and 100 μl of each sample was plated on a separate culture medium and cultured at 28 ° C for a certain period of time. The culture conditions were observed periodically during the culture period, and the cultured colonies were inoculated on a fresh medium to isolate single colonies. The colonies were selected by observing with a microscope. The culture broth and culture conditions were as follows (Table 1).

The strains isolated from each strain were cultured on an agar medium. Cells were collected, suspended in 20% sterilized glycerol, and stored in a -70 ° C ultra-low temperature refrigerator. The isolated strains were cultured in a solid medium, suspended in sterilized 10% skim milk, lyophilized and stored at 4 ° C or below.

Actinomycetes Separation medium  And culture conditions microbe Separation medium and composition pH Incubation temperature Incubation period


Actinomycetes HV agar (g / l)
Humic acid 1
Na ₂ HPO ₄ 0.5
KCl 1.71
MgSO ₄ 7H ₂ O 0.05
· FeSO ₄ 7H ₂ O 0.01
· CaCO ₃ 0.02
· Agar 18


pH 7.2


28 ℃


7-30 days

< Example  2> Identification of the strain

For the identification of the strains isolated in Example 1, 16S rRNA gene sequencing was performed.

Specifically, colony PCR was performed to obtain the nucleotide sequence of the 16S rRNA gene of the strain. From a single colony, a toothpick was picked and mixed with PCR-premix (iNtRON Biotechnology, Korea), and 16S rRNA gene was amplified using 27f and 1492r primers. The amplified PCR products were purified using a Wizard PCR prep kit (Promega, Medison, Wis., USA), and the nucleotide sequences were obtained from Daejon, Korea. The nucleotide sequence was compared with the GenBank database using NCBI BLSAT. We aligned the nucleotide sequence with CLUSTAL X and created a neighbor-joining tree using PHYDIT program version 3.0. The nucleotide sequence of the 16S rRNA gene of the strain (SEQ ID NO: 1) was analyzed based on the neighb-joining algorithm.

As a result, it was found that this strain was a novel strain showing a homology of about 99.7% with Streptomyces scopoluridis RB72T (Fig. 1). The strains used in the present invention include Streptomyces scopoluridis scopuliridis ). The strain of Streptomyces Scott Lee pulley disk (Streptomyces scopuliridis ) KR-001 and deposited it with the deposit number KCTC 12156BP in the Life Resource Center of the Korea Research Institute of Bioscience and Biotechnology on Mar. 09,

< Example  3> Streptomyces Scotopuridis KR -001 concentration of strain culture medium When processing foliage  Active measurement

In order to measure the activity of the culture broth of Streptomyces scorpionis strain KR-001 obtained from <Example 1> upon foliar treatment, the following experiment was conducted.

Specifically, 500 ml of a Bennet liquid medium was placed in a 500 ml baffled flask, and 1 ml of the pre-cultured medium was inoculated and cultured at 27 占 폚 and 180 rpm for 7 days. Streptomyces scopoliliis KR -001 was diluted to a concentration of 1, 1/2, and 1/4 (including 0.1% Tween-20), and weeds (weevils, sorghum, The leaves were treated with 14 ml of pot per pot, and 5 days after the application, the symptoms and the efficacy of the extracts were assessed by the questionnaire survey. The composition components of the Bennett medium are shown in Table 2 below.

As a result, as shown in Fig. 2 and Table 3, the grazing power for the eight cultivars in the culture broth was 90% to 100%, and 70% to 100% even at 1/2 concentration. Among the weed species, weeds were relatively strongly expressed in the weevils, lemurs, and mullets. The main symptom was burndown, and at 1/4 concentration, it was confirmed that some of herbaceous plants and herbaceous plants turned violet (see Tables 3 and 2) ).

Bennett  Composition of the medium Composition component g / L Glucose 10 Yeast extract One Peptone 2 Beef extract One

In the greenhouse condition, three kinds of phloem and weeds (barren, sorghum, phe) and five species Light  Weeds (Lawn, The , Doodle, meadow) Streptomyces Scotopuridis KR -001 by the dilution ratio of culture medium When processing foliage  activation(%) Rate
(folds) Fertility (%) wire grass Sorghum Peck Mad Japonica The A toddler convolvulus One 100 90 95 100 100 100 100 95 1/2 100 80 70 100 100 70 90 80 1/4 95 50 20 80 100 50 70 20

DIGSA, Digotaris sanguinalis ), sorghum (SORBI, Sorghum bicolor , dorsal (ECGCG, Echinochloa crus - galli ), blackheaded (SOLNI, Solanum nigrum), Aeschynomene indica (AESIN, Aeschynomeme indica , ABUTH, Abutilon avicennae ), dogs (XANSI, Xanthium strumarium ), meadow (CAGEH, Calystegia japonica )

< Example  4> Streptomyces Scotopuridis KR -001 Solvent of culture medium By fraction  Activity measurement in foliar treatment

Experiments were carried out as follows to measure the killing power of the solvent fraction of the culture solution obtained from the <Example 3>.

Specifically, the cultured strain of Streptomyces scopolridis KR-001 was fractionated into hexane, ethyl acetate, butanol and water. Each solvent fraction by targeting indica ^{8 μg mL -1, 16 μg mL} -1, 32 μg mL -1, 63 μg mL -1, 125 μg mL -1, 250 μg mL -1 and 500 μg mL ^-1 After 5 days of foliar treatment, the females were assessed for appearance and medicinal efficacy according to the criterion table.

As a result, as shown in FIG. 3 and Table 4, no activity was observed in the hexane and water layers, and the activity was expressed in the ethyl acetate and butanol layers. Especially, the strongest acetic acid was found in the ethyl acetate layer. Ethyl acetate was measured at a concentration of 8 μg mL ^-1 , 16 μg mL ^-1 , 32 μg mL ^-1 , 63 μg mL ^-1 , 125 μg mL ^-1 , 250 μg mL ^-1 and 500 μg mL ^-1 , It was confirmed that strong supercritical force was shown at 80%, 90%, 95%, 100%, 100%, 100% and 100% respectively. At this time, it was confirmed that the yield of the active material for each solvent fraction was 0.03% for the hexane fraction, 3.61% for the ethyl acetate fraction, 17.84% for the butanol fraction, and 73.67% for the water fraction (Table 4 and FIG. 3).

For greenhouse conditions in greenhouse conditions Streptomyces Scotopuridis KR -001 Solvent of culture medium By fraction When processing foliage  activation(%) Solvent layer Fertility (%) 500 (μg mL ^-1 ) 250 125 63 32 16 8 Hexane 0 0 0 0 0 0 0 Ethyl acetate 100 100 100 100 95 90 80 Butanol 80 70 50 20 0 0 0 water 0 0 0 0 0 0 0

< Example  5> Streptomyces Scotopuridis KR -001 of the culture broth Fraction  Effect measurement in soil treatment

In order to measure the soil treatment effect of the fraction of the culture broth of the present invention obtained from <Example 4>, the following experiment was conducted.

Specifically, the plants were sowed with four kinds of weevils and weeds (barkworm, sorghum, dandelion, and canola) and 5 kinds of light leaf weeds (lambs, , Light / dark = 14/10 hours), the ethyl acetate fraction of the culture broth of Streptomyces scopuliriides KR-001 strain was treated with 2000 μg mL ^-1 , 1000 μg mL ^-1 , 500 μg mL ^-1 , 250 μg mL ^-1 , and 125 μg mL ^-1 , respectively. After 15 days of treatment, the symptom and drug efficacy were investigated by scintillation (0% to 100%) according to the criteria of weakness.

As a result, as shown in Fig. 4 and Table 5, the grafting power of the target weed species at a concentration of 2000 μg mL ^-1 in the ethyl acetate fraction was 95% to 100%, and even at a concentration of 1000 μg mL ^-1 , (100%) against all other species. At the concentration of 2000 μg mL ^-1 and 1000 μg mL ^-1 , the emergence of the seedlings and grasses was observed but the other shoots did not appear at the ground level. This phenomenon was observed after each weed seed germination, Acetate fractions. In the test specimens, the barnyardgrass, wheatgrass, ramie, toucorpion, and meadow were completely dead at 500 μg mL ^-1 concentration, and 250 μg mL ^{-1 of} canola and 125 μg mL ^{-1 of} cane flower were completely dead, (FIGS. 4 and 5).

Four kinds of phloem and weeds (green bean, sorghum, coriander, dense) in greenhouse condition and five species Light  Weeds (Lawn, The , Cockleys, meadow flowers) Streptomyces Scopuliri disrespect KR -001 Effect of Soil Treatment of Ethyl Acetate Fraction density
(μg mL ^-1 ) Fertility (%) wire grass Sorghum Peck couch grass Mad Japonica The A toddler convolvulus 2000 100 95 100 100 100 100 95 100 100 1000 100 95 100 100 100 100 90 100 100 500 100 20 70 100 100 40 40 100 100 250 90 0 0 100 70 0 30 80 100 125 50 0 0 50 60 0 0 0 100 62.5 0 0 0 0 0 0 0 0 0

< Example  6> Streptomyces Scotopuridis KR -001 strain culture medium Fraction When processing foliage  Active measurement

In the foliar treatment of the fraction of the strain of the present invention obtained from <Example 4> In order to measure the activity, the following experiment was carried out.

Specifically, four kinds of weevils and weeds (barnacles, sorghum, dandelion, and weevil) and five kinds of light leaf weeds (Lawn, Zebra, Zebra, Tadomari, and Japanese Meadow) were sowed and grown for 9 days in a greenhouse condition. The ethyl acetate fraction of the strain culture of the present invention obtained from Example 4 was diluted to 2000 μg mL ^-1 , 1000 μg mL ^-1 , 500 μg mL ^{-1 with} 50% acetone and Tween-20 0.1% ^{250 μg mL -1, 125 μg mL} -1, 62.5 μg mL was diluted to prepare foliar spray treatment so that the concentration of ^-1, for 12 days after the appearance of symptoms and the efficacy dalgwan by the weak standard list irradiation (0% to 100% ).

As a result, as shown in Fig. 5 and Table 6, the vigorous force against nine weeds at the treatment concentrations of 2000 μg mL ^-1 and 1000 μg mL ^-1 showed a strong activity of 95% to 100%, and 500 μg mL &lt; ^-1 &gt; In the early stage of the drug treatment, the external symptoms were manifested and the effect was increased with the elapse of the treatment date, and it was confirmed that the survival persistence was maintained even after 10 days of drug treatment. The main symptom was the formation of a needle bed at the beginning of the treatment and then the test was decayed over time and eventually decanted. At the concentration of 250 μg mL ^-1 or less, it was confirmed that the strong kernels were stronger depending on the species, while the kennels, lemurs, and lepidoptera showed 95% or higher kernels at the concentration of 62.5 μg mL ^-1 (FIGS. 5 and 6).

Four kinds of phloem and weeds (green bean, sorghum, coriander, dense) in greenhouse condition and five species Light  Weeds (Lawn, The , Doxycycline, meadow flowers) in the ethyl acetate fraction When processing foliage  activation density
(μg mL ^-1 ) Fertility (%) wire grass Sorghum Peck couch grass Mad Japonica The A toddler convolvulus 2000 95 100 100 100 100 100 100 100 100 1000 95 100 95 100 100 100 100 100 100 500 90 80 100 100 100 100 95 100 100 250 80 100 60 90 100 100 70 100 100 125 50 100 40 90 100 100 40 90 100 62.5 40 95 20 80 95 100 30 90 70

< Example  7> Streptomyces Scotopuridis KR -001 strain culture medium Fraction  Visibility in Greenhouse Conditions Sicyos angulatus ) For Foliar treatment  Check control effect

The following experiment was carried out in order to confirm the effect of the fraction of the strain of the present invention obtained from the above <Example 4> on the control of the foliage on the Sycheos angulatus in the greenhouse condition.

Specifically, the ethyl acetate fraction was subjected to foliar treatment at a concentration of 2000 μg mL ^-1 and 1000 μg mL ^-1 , followed by irradiation at 7 days.

As a result, as shown in Figure 6, after the foliar treatment, and 24 over time begins to outward symptoms are expressed as by 5 days over 2000 μg mL ^- to both the ¹ and 1000 μg mL ^-1 concentration visible foil completely (Fig. 6).

< Example  8> Streptomyces Scotopuridis KR -001 of the culture broth Fraction  Through in-vivo implementation in greenhouse conditions Thorny night  Check control effect

The following experiment was conducted to confirm the effect of the fraction of the strain of the present invention obtained from <Example 4> in the greenhouse condition through in vivo transfer of the fraction of the strain of the present invention.

Specifically, when the three leaves were expanded in the greenhouse condition, the prepared ethyl acetate fraction was soaked in gauze at a concentration of 1000 μg mL ^-1 on the trilobal stem, sealed with aluminum foil, and treated locally , And allowed to stand for 24 hours under dark conditions to be sufficiently absorbed into the plant. After 24 hours of treatment, the gauze and aluminum foil were removed and maintained in the same greenhouse condition, observing daily progress, and then irradiated 6 days after treatment.

As a result, as shown in Fig. 7, appearance of symptoms began to appear after 2 days from the local treatment, and 6 days later, the untreated spikelets developed to 7 leaves, but in the treated spikes only 4 leaves were developed In addition, it can be confirmed that the test is proceeding from the periphery of the fourth leaf and the second leaf. Thus, it was confirmed that the ethyl acetate fraction was simultaneously transferred upward and downward (FIG. 7).

< Example  9> Streptomyces Scotopuridis KR -001 of the culture broth Fraction  Under packaging conditions On a spike  About When processing foliage  Check control effect

The following experiment was conducted in order to confirm the effect of controlling the foliar treatment on the visibility of the fraction of the strain of the present invention obtained from <Example 4> under the packaging conditions.

Specifically, the ethyl acetate fraction of the strain culture of the present invention obtained from the above <Example 4> was confirmed to have a strong controlling effect in a greenhouse condition against the environmental pest which was designated as "ecosystem disturbed wild plant" At the same time, field application evaluation was conducted under actual packaging conditions. The test site was located on the southern part of the riverside in front of the village hall of Andrews Village, 31-15 Degunde, Gangwon-gun, Gyeonggi-do. The testing period was from September 22, 2011 to October 06, 2011. During the treatment, the growth conditions of spikelet were 10 to 15 weeks, and the length was 2 m to 3 m. The treatment area was 1 m × 1 m and treated with 300 mL of each concentration. The final concentration of the ethyl acetate fraction was weighed to a concentration of 2000 μg mL ^-1 and 4000 μg mL ^-1 and dissolved in a small amount of acetone and diluted with a pharmaceutical preparation (containing 50% acetone and 0.1% Tween-20) , 5 days, 8 days and 14 days after treatment.

As a result, as shown in Fig. 8 and Table 7, the extractive force against the visible layer in the ethyl acetate fraction of the culture broth of the present invention was improved over time after the treatment with the agent, The inhibitory effect on visibility was 50%, 90% and 100% at 5, 8 and 14 days after treatment at 4000 μg mL ^-1 , respectively. The control effect at 2000 μg mL ^-1 was 40% 70% and 80%, respectively. Generally, herbicidal active materials derived from natural materials are rapidly effective, so that the efficacy of the herbicidal active substance is manifested within a few hours of the treatment, and it is known that the active ingredient is regenerated after a lapse of a certain period of time after the treatment, The survival rate was maintained even after one week, and it was confirmed that the sustained action was excellent. In addition, when the stem located at the lower part of the treated leaf was broken like a mosaic disease, it was judged that the drug migrated to the lower stem as well as the treated leaf (FIGS. 8 and 7)

Under packaging conditions On a spike  About Streptomyces Scotopuridis KR -001 Culture of the strain Ethyl acetate fraction Foliar treatment  effect density
(μg mL ^-1 ) Fertility (%) 5 DAT 8 DAT 14 DAT 4000 50 90 100 2000 40 70 80

DAT: Days after treatment (days after treatment)

< Example  10> Streptomyces Scotopuridis KR -001 strain culture medium Fraction heating For our weeds (hornblende, wormwood, hornblende, clover) When processing foliage  Control effect

In order to confirm the control effect on the weeds which are difficult to control, such as hornblende, wormwood, rhizome or clover, under the packaging conditions of the fraction of the strain of the present invention obtained from <Example 4>, the following experiment was carried out .

Specifically, the test was conducted in the vicinity of the Korea Chemical Research Institute, Yuseong-gu, Daejeon Metropolitan City, and the test area was selected from October 4, 2011 to October 16, 2011. The treatment compartment was treated with 200 ml of each concentration at 1 m × 1 m. The final treatment concentration of the ethyl acetate fraction of the strain culture medium of the present invention obtained from <Example 4> was weighed to a concentration of 2000 μg mL ^-1 and 4000 μg mL ^-1 and dissolved in a small amount of acetone, 50% acetone, and 0.1% Tween-20). After clotting, the clover samples were examined at 5, 10, and 15 days after the treatment, 5, 8, and 10 days after the mugwort, and 8, 12 days after the clover treatment.

 <10-1> Horseshoe vine Humulus japonicus ) Check the effectiveness of the control

9 and as shown in Table 8, the ethyl acetate fraction of a strain culture solution of the present invention obtained from the <Example 4> is Humulus japonicus (Humulus japonicus ), the control effect was about 40% and 30% after 5 days of treatment at the concentrations of 2000 μg mL ^-1 and 4000 μg mL ^-1 , respectively, 100% and 95%, respectively, and 100% after 15 days, respectively (Table 8 and FIG. 9). It was confirmed that the sustained action was sustained until the 15th day after the treatment, and the sustained action was sustained.

Hwangsam vine in packaging condition Humulus japonicus ), Wormwood Artemisia princeps ), Equisetum arvense ) And clover ( Trifolium repens )on  The ethyl acetate fraction of Foliar treatment  effect density
(μg mL ^-1 ) Fertility (%) Horseshoe Mugwort Pecker clover 4000 100 100 100 100 2000 100 100 100

<10-2> Mugwort Artemisia princeps ) Check the effectiveness of the control

As shown in Figure 10 and Table 8, the ethyl acetate fraction of the <Example 4> A strain culture solution of the present invention is obtained from mugwort (Artemisia princeps ), but showed complete control against mugwort at concentrations of 2000 μg mL ^-1 and 4000 μg mL ^{-1. After} 3 days of treatment, the effect began to develop, and after 5 days, 4000 μg mL ^-1 and 80% at the concentration of 2000 μg mL ^-1 . After 8 days, both concentrations were 100%, indicating that the supernatant was maintained (see Tables 8 and 10 ).

<10-3> Rape ( Equisetum arvense ) Check the effectiveness of the control

As shown in Figure 11 and Table 8, the ethyl acetate fraction of a strain culture solution of the present invention obtained from the <Example 4> is Equisetum arvense (Equisetum arvense . The efficacy was very rapid, and 95% and 90% of the herbicidal activity was observed at 4000 μg mL ^-1 and 2000 μg mL ^-1 after 5 days of treatment, respectively. After 8 days, (Table 8 and Fig. 11).

<10-4> Clover ( Trifolium repens ) Check the effectiveness of the control

As shown in Fig. 12 and Table 8, the ethyl acetate fraction of a strain culture solution of the present invention obtained from the <Example 4> is clover (Trifolium repens ). After 4 days of treatment, spots were formed on the leaves. After 8 days, the leaves were completely damaged. After 12 days, the leaves were streaked to the stem, (Table 8 and Fig. 12).

< Example  11> Streptomyces Scotopuridis KR -001 strain culture medium Fraction  Evaluation of crop selectivity in greenhouse conditions

In order to evaluate the crop selectivity of the fraction of the culture broth of the present invention obtained from <Example 4>, the following experiment was conducted.

Specifically, crop selectivity of the ethyl acetate fraction of the strain culture of the present invention obtained from the above Example 4, which confirmed excellent grazing power for various kinds of weeds including visible leaves, was confirmed. The target crops were 3 kinds of crops such as rice, wheat and barley, 2 kinds of crops such as pepper and tomato and 2 kinds of crops such as pepper and tomato. At the time of treatment, the growth condition of each crop was 3.5 leaf of rice, 1.2 leaf of wheat, , And tomato was 4.4 leaf. The treatment concentrations were 4000 μg mL ^-1 , 2000 μg mL ^-1 , 1000 μg mL ^-1 , 500 μg mL ^-1 , 250 μg mL ^-1, and 125 μg mL ^-1 , respectively. After 14 days of treatment, And examined with a naked eye.

As a result, as shown in Fig. 13 and Table 9, it was confirmed that all of the target crops were susceptible to the attack of weakness, and that there was no crop selectivity. In particular, tomatoes were most severely attacked by pepper and tomatoes. In the case of tomatoes, 100% of the total concentration of the tomatoes was induced, and the severity was the worst. In the pepper, 70% to 100% . In the case of barley, the concentration of the barley was 50% to 80%, while that of rice and wheat was lower than 30%, which was relatively low. Therefore, it has been confirmed that the herbicidal active material derived from actinomycetes should be used non-selectively because there is no crop selectivity (Fig. 13 and Table 9).

Three kinds of plants and crops such as rice, wheat and barley in a greenhouse condition, pepper and tomato Light  For two crops Streptomyces Scotopuridis KR -001 &lt; / RTI &gt; of the ethyl acetate fraction of the culture broth crops Weak (%) 4000 (μg mL ^-1 ) 2000 1000 500 250 125 rice plant 30 30 10 10 5 0 wheat 30 30 30 20 20 20 barley 80 80 80 70 60 50 pepper 90 90 90 90 80 70 tomato 100 100 100 100 100 100

< Example  12> Streptomyces Scotopuridis KR -001 strain culture medium Fraction  Of the control compound Live  compare

In order to confirm the viability of the fraction of the culture obtained from the above <Example 4>, the binding forces of Bialaphos and Glufosinate were compared with each other.

Specifically, after planting soil in a plastic square pot with a surface area of 350 cm ² , four kinds of weeds including weevils and four weeds and three weedy light weeds including weevils were sowed, respectively, and greenhouse conditions (25 ± 3 ° C, 14 / 10h = Light / dark), 15 days after sowing, the ethyl acetate fraction of the culture broth, Bialaphos and Glufosinate were treated with 250, 500 and 1000 μg of drug preparation solution (containing 50% acetone and 0.1% Tween-20) mL ^{-1, and} treated with 14 ml / pot of foliar application.

As a result, as shown in FIG. 14, the ethyl acetate fraction, Bialaphos, and Glufosinate of the strain culture medium of the present invention obtained from the <Example 4> were 250 μg mL ^-1 , 500 check plants showed an μg mL ^-1 and 1000 μg mL ^-1 of comparable concentration levels in live choryeok were (Fig. 14). Therefore, it is considered that the extractive force of the ethyl acetate fraction of the culture broth of the present invention is similar to the control compound Bialaphos or Glufosinate.

< Example  13> Streptomyces Scotopuridis KR -001 of the culture broth Fraction  Identification of herbicidal active substances

In order to confirm the herbicidal activity of the fractions of the culture broth of the present invention obtained from <Example 4>, the following experiment was conducted.

Specifically, the ethyl acetate fraction of the culture broth of the present invention obtained from <Example 4> was subjected to HPLC. The analysis conditions are as follows (Table 10).

As a result, Rt. Peak was confirmed at 6 minutes 50 seconds, 9 minutes 10 seconds, and 15 minutes 30 seconds (Fig. 15).

Streptomyces Scotopuridis KR HPLC analysis of ethyl acetate fraction of culture broth Solvent 40% MeOH Column C-18 Temperature 40 ℃ Flow 1 ml / min UV 254 nm

< Example  14> Streptomyces Scotopuridis KR -001 &lt; / RTI &gt; Live  Confirm

The Rt. 6 minutes 50 seconds, 9 minutes 10 seconds, and 15 minutes 30 seconds, and the results of the experiments were carried out as follows.

Specifically, Rt. &Lt; RTI ID = 0.0 > 6 min 50 sec, 9 min 10 sec and 15 min 30 sec were subjected to Prep-HPLC under the above analytical conditions (Table 10) to give fr. 1 - 7. Each fraction was subjected to foliar treatment at a concentration of 112.5 μg mL ^-1 , 225 μg mL ^-1 , 450 μg mL ^-1 and 900 μg mL ^-1 , and after 5 days, The results of this study are as follows.

As a result, as shown in Fig. 16, fr. 3, 4 and 5, respectively. Fr. 3, 4 and 5 were isolated and purified. 6 min 50 sec, 9 min 10 sec and 15 min 30 sec were identified as Herbicidin B, Herbicidin A and Herbicidin F ( The Journal of Antibiotics 1982,35 1711-1714.) (Fig. 16).

Korea Life Science Research Institute KCTC12156BP 20120309

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Claims (10)

Deposited as accession number of KCTC 12156BP Streptomyces Scott Lee pulley disk (Streptomyces scopuliridis ) KR-001 strain.
A culture medium of Streptomyces scopoliliidis KR-001 deposited with the deposit number KCTK 12156BP, a culture thereof, a fraction of the culture medium and an active fraction of the fraction, as an active ingredient, Wherein the fraction is fractionated sequentially using hexane, ethyl acetate, butanol and water as a solvent.
delete The method according to claim 2, wherein the active fraction is a fraction eluted at 6 to 7 minutes, 8 to 10 minutes, or 15 to 20 minutes using methanol, ethanol, or an aqueous solution thereof in a column chromatography, By weight of the herbicidal composition.
3. The herbicidal composition for controlling weeds according to claim 2, wherein the weeds are weed and weeds, weed weeds or weed weeds.
The method of claim 5, wherein the weeds are hwabongwa indica (Digotaris sanguinalis ), sorghum ( Sorghum bicolor , Agropyron amithii ) and Echinochloa wherein the herbicidal composition is selected from the group consisting of crus - galli .
6. The method of claim 5, wherein the weed weeds are selected from the group consisting of Solanum nigrum), Aeschynomene indica (Aeschynomeme indica , Abutilon avicennae , Xanthium strumarium ) and meadows ( Calystegia japonica ). &lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
6. The method according to claim 5, wherein the heating weed is a syrup angulatus , Humulus japonicus ), wormwood ( Artemisia princeps , equisetum arvense ) and clover ( Trifolium repens ). &lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
The seeds or their habitats of a strain selected from the group consisting of Streptomyces scopoliliidis KR-001 strain deposited with the deposit number KCTC 12156BP, a culture thereof, a fraction thereof, and an active fraction of the fraction, Lt; RTI ID = 0.0 &gt; weed control &lt; / RTI &gt;
10. The method for controlling weeds according to claim 9, wherein the weeds are weed and weeds, weed weeds or weed weeds.

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