KR102523179B1 - Composition for controlling weeds comprising Streptomyces sp. mutant KRA16-334M70 or a culture thereof - Google Patents

Abstract

The present invention relates to a composition containing the Streptomyces sp. mutant strain KRA16-334M70 or a culture medium thereof. Specifically, the herbicidal activity can be greatly improved by adding an auxiliary to the Streptomyces sp. mutant strain KRA16-334M70 or the culture medium thereof, so that weed control can be carried out effectively by using the same.

Description

Composition for controlling weeds comprising Streptomyces genus mutant KRA16-334M70 or a culture solution thereof {Composition for controlling weeds comprising Streptomyces sp. mutant KRA16-334M70 or a culture thereof}

The present invention relates to a composition for controlling weeds comprising Streptomyces genus mutant strain KRA16-334M70 or its culture medium, and more particularly, by adding an adjuvant to Streptomyces genus mutant strain KRA16-334M70 or its culture medium, the herbicidal activity is It relates to a greatly improved composition for controlling weeds.

Weeds or weeds are not absolute, but relative concepts, and they are plants that are not appropriate for the time and place, which occurred when humans started agricultural life. Weeds are major control targets that greatly reduce productivity and marketability by competing with and deodorizing light, nutrients, moisture, etc. in crop cultivation, mediating pests and making agricultural work difficult. Weeds occur throughout the year regardless of location, season, environment, etc., and a lot of effort and money are required to control them. In addition, since there are many types of weeds and they are diverse, they are divided into flower and weeds, broad-leaved weeds, and weeds according to plant taxonomic classification and morphological classification, and divided into annual weeds, monthly weeds and perennial weeds according to life type. Therefore, in general, weeds are classified into flowering plants, annual weeds, perennial weeds, broad-leaved annual weeds, perennial weeds, and panicle annual weeds, perennial weeds, etc., or conversely, annual flowers and weeds, broad-leaved weeds, panicle weeds, or perennial flowers and weeds, broad-leaved weeds, and fireflies weeds. separated by

The stems of grasses and weeds have well-distinguished nodes and internodes, and leaves grow alternately from the nodes, and the leaves are divided into leaf sheaths and leaf blades that surround and protect the stems. The leaf body is narrow and long, and the leaf veins are formed in parallel. Blood, indica, foxtail, foxtail, reed, and silver grass belong to this category. Weeds belonging to the Sedges family have similarities with Sedges, but most of them are distinguished by the fact that the cross section of the stem is triangular and there is no ligule or leaf auricle. The leaves are narrow and ridged, with pointed tips, and a small number of small flowers. These include cockroaches, spiny spinners, olbanggae, barnacles, and tadpole gorillas. A broad leaf weed is a plant that does not belong to the herbaceous weeds or weeds of the family Dipperaceae, and is literally a weed with relatively wide leaves. The leaves are mainly elliptical, ovate, and lanceolate, and the leaf veins are intertwined like a net. Many weeds such as egret, shamrock, mugwort, shepherd's purse, amaranth, waterweed, sputum, and viburnum, which are common around us, belong to this category.

Currently, various organic synthetic herbicides exhibiting high herbicidal activity at low cost are used for weed control, but the emergence of resistant weeds, environmental harm and residual toxicity are pointed out as problems. In particular, the emergence of resistant weeds due to the long-term use of synthetic organic herbicides is recognized as a serious socio-economic problem (Riches et al. 1996).

In order to overcome and replace problems such as strong toxicity of synthetic organic herbicides, persistence in soil, and emergence of resistant varieties, research to develop herbicidal active substances with new mechanisms of action and new structures from microbial metabolites is being actively conducted. Herbicidal active substances such as anisomycin, bialaphos, cycloheximide, herbimycin, and toyocamycin were discovered from actinomycetes of the genus Streptomyces sp. It became. However, microorganisms that form hyphae, such as actinomycetes, have many difficulties in designing bioreactors and optimizing fermentation processes due to their unique morphology and demanding physiology of secondary metabolism, especially due to low productivity of secondary metabolites. Industrialization is difficult due to economic problems.

The productivity of actinomycete secondary metabolites can be improved through optimization of culture conditions including strain improvement, and high-productivity strain development and fermentation process technology are recognized as the most important technologies that determine the know-how and economic feasibility of the agro-biological industry. Mutagenesis is one of the most reliable and widely used techniques for strain improvement to increase the yield of secondary metabolites of actinomycetes. UV rays, X-rays, γ-rays, lasers, neutrons, and chemiphoresis are used as physical methods for mutagenesis, and methyl methane sulfonate (MMS), hydroxylamine (HA), and N-methyl- Chemicals such as N-nitro-N-nitrosoguanidine (MNNG) are used. Among them, the physical method for obtaining broad-spectrum mutations is through ultraviolet (UV) irradiation, which is safer than chemical mutagenesis and can obtain improved secondary metabolite production from industrial microbial strains through random mutagenesis and fermentation screening. there is.

Accordingly, the present inventors performed strain improvement through mutagenesis on Streptomyces sp. , It was confirmed that the productivity of the herbicidal active substance 334-HP50-W4 (TMC-86A) contained in this strain or its culture medium was improved, and also the present invention was completed by developing an adjuvant that enhances the herbicidal activity of the composition.

Republic of Korea Patent Registration No. 10-2010471 (2019.08.13. Notice) Republic of Korea Patent Registration No. 10-2032913 (2019.10.16. Notice)

An object of the present invention is to provide a composition for controlling weeds comprising Streptomyces genus mutant strain KRA16-334M70 or its culture medium.

Another object of the present invention is to provide a composition for controlling weeds comprising Streptomyces genus mutant strain KRA16-334M70 or its culture medium with enhanced herbicidal activity, and an adjuvant.

The problem to be solved by the present invention is not limited to the above-mentioned problem (s), and another problem (s) not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a composition for controlling weeds comprising Streptomyces sp. mutant KRA16-334M70 strain (KCTC15096BP) or its culture medium as an active ingredient.

The strain may have an increased yield of herbicidal active substances compared to Streptomyces sp. strain KRA16-334 (KCTC13357BP).

The composition may include a substance represented by Formula 1 below as a herbicidal active substance.

[Formula 1]

The composition may further include an adjuvant.

The adjuvant may be at least one selected from the group consisting of castor oil polyoxyethylene ether, secondary alcohol ethoxylate, and polyoxyethylene lauryl ether. there is.

The adjuvant may be included in an amount of 0.01 to 0.5% (v/v) based on the total amount of the composition.

The culture medium of the strain may be diluted 1 to 32 times.

The composition may have control activity against flowering plants and weeds or broadleaf weeds.

The composition is one or two selected from the group consisting of Sorghum bicolor , Echinochloa crus-gall i, Indica ( Digitaria sanguinalis ), Agropyron smithii , and Panicum dichotomiflorum as flowers and weeds. It may have weed control activity for more than one species of plant.

The composition is a weed for one or two or more plants selected from the group consisting of Solanum nigrum , Aeschynomene indica , Abutilon avicennae , Xanthium strumarium and Calystegia japonica as broadleaf weeds. It may have inhibitory activity.

In addition, in order to solve the above problems, the present invention Streptomyces ( Streptomyces sp.) Mutant strain KRA16-334M70 strain (KCTC15096BP) or a culture medium thereof to all or part of weeds, their seeds or the step of treating their habitat Including, it provides a weed control method.

The weeds may be herbaceous weeds or broadleaf weeds.

According to the present invention, since the herbicidal activity can be greatly improved by adding an adjuvant to the Streptomyces genus mutant strain KRA16-334M70 or its culture medium, weed control can be effectively performed using this.

Figure 1 shows the UV mutation method and conditions used for the Streptomyces genus mutant strain KRA16-334M70 according to an embodiment of the present invention.
Figure 2a is a result of high-performance liquid chromatography (HPLC) analysis of the herbicidal active substance 334-HP100-M produced by the Streptomyces genus mutant strain KRA16-334M70 strain according to an embodiment of the present invention, Figure 2b is the herbicidal activity It shows a graph of quantitative analysis of material 334-HP100-M.
Figure 3a is a high-performance liquid chromatography (HPLC) analysis result of the herbicidal active substance 334-HP50-W4 produced by the Streptomyces genus mutant strain KRA16-334M70 strain according to an embodiment of the present invention, Figure 3b is the herbicidal activity A graph of quantitative analysis of material 334-HP50-W4 is shown.
Figure 4 is a photograph evaluating the herbicidal activity of mutant candidate strains of the genus Streptomyces according to an embodiment of the present invention.
Figure 5 compares the yield of herbicidal active substances produced by mutant candidate strains of the genus Streptomyces according to an embodiment of the present invention.
Figure 6a is a graph showing the herbicidal activity according to the concentration in Streptomyces genus mutant KRA16-334M70 strain culture filtrate alone according to an embodiment of the present invention, and Figure 6b is the KRA16-334M70 It is a graph showing the herbicidal activity according to the concentration in indica and crow when treated by mixing Tween-20 (0.1%) with the strain culture filtrate.
Figure 7 is a photograph comparing the results 10 days after treatment with each of the three kinds of adjuvants, the M70 strain culture medium and the M70 strain culture medium + three kinds of adjuvants, respectively, to the control group according to an embodiment of the present invention.
Figure 8 is a photograph comparing the results 4 days after treatment with each of the three adjuvants in a 2, 4, 8, 16, and 32-fold dilution of the M70 strain culture medium according to an embodiment of the present invention to the control group.

Before describing the present invention in detail, the terms or words used in this specification should not be construed as unconditionally limited in a conventional or dictionary sense, and in order for the inventor of the present invention to explain his/her invention in the best way It should be noted that concepts of various terms may be appropriately defined and used, and furthermore, these terms or words should be interpreted as meanings and concepts corresponding to the technical idea of the present invention.

That is, the terms used in this specification are only used to describe preferred embodiments of the present invention, and are not intended to specifically limit the contents of the present invention, and these terms represent various possibilities of the present invention. It should be noted that it is a defined term.

In addition, in this specification, it should be noted that singular expressions may include plural expressions unless the context clearly indicates otherwise, and similarly, even if they are expressed in plural numbers, they may include singular meanings. do.

Throughout this specification, when a component is described as "including" another component, it does not exclude any other component, but further includes any other component, unless otherwise stated. It can mean you can do it.

In addition, in the following description of the present invention, a detailed description of a configuration that is determined to unnecessarily obscure the subject matter of the present invention, for example, the known technology including the prior art, may be omitted.

Hereinafter, the present invention will be described in more detail.

The present invention provides a composition for controlling weeds comprising a Streptomyces sp. strain KRA16-334M70 mutant or a culture solution thereof deposited under accession number KCTC15096BP.

Streptomyces genus mutant strain KRA16-334M70 strain according to the present invention [hereinafter referred to as 'KRA16-334M70 strain', 'strain KRA16-334M70', 'strain M70', 'M70 strain' or 'mutant strain M70'] is Streptomyces drozdowiczii KRA16-334 strain (accession number KCTC 13357BP) identified through isolation and culture from forest soil around Pungsan-ri, Boryeong-gun, Chungcheongnam-do was mutated and improved.

The present inventors named the novel strain as Streptomyces sp. KRA16-334M70 ( Streptomyces sp. KRA16-334M70) strain and deposited it at the Korea Research Institute of Bioscience and Biotechnology (KCTC) on September 22, 2022, accession number KCTC Granted 15096 BP.

The Streptomyces genus mutant KRA16-334M70 strain or its culture medium is characterized in that it has weed control activity.

The culture solution is a result of culturing a microbial strain in a medium and may include metabolites of the microorganism. The culture medium may be a liquid medium in which the strain is cultured, but is not limited thereto, and the strain may be cultured in a conventional microbial culture medium. In addition, the culture solution may be a culture solution in which the strain is cultured or a culture filtrate obtained by physically filtering the culture solution, and the culture solution may be undiluted or diluted to treat weeds. As long as the culture broth exhibits herbicidal activity against weeds, it can be treated with weeds through treatment processes such as filtration, concentration, and dilution.

The weed control activity reduces plant growth or leads to death due to serious damage such as leaf yellowing and blight, and is used in the present invention in the same sense as herbicidal activity or herbicidal activity.

The mutant KRA16-334M70 strain may have an increased yield of herbicidal active substances compared to its wild-type strain, KRA16-334 (KCTC13357BP), and the yield may be increased by 2 or more times or 3 times or more.

In a specific embodiment of the present invention, in order to increase the yield of the herbicidal active substance of the wild-type strain KRA16-334, Streptomyces drozdowicchi KRA16-334 wild-type strain is firstly irradiated with ultraviolet rays to induce mutation, and secondary Mutagenesis was induced by treatment with chemical substances, and strains exhibiting the highest herbicidal activity and yield of herbicidal active substances were selected among the mutant candidate strains. As a result, the mutant strain M70 showed a 10-fold or more improvement in herbicidal activity compared to the control group (FIG. 4), and a 4-fold increase in the yield of the herbicidal active substance 334-W4 compared to the wild type (FIG. 5).

The composition may include a material represented by Chemical Formula 1 below or herboxidiene as an active material.

[Formula 1]

The herbicidal active substance of the wild-type strain KRA16-334 is a substance represented by Formula 1 [334-HP50-W4, 334-W4 or TMC-86A] or herboxidiene [334-HP100-M or 334-M100 ], which is described in Patent Registration No. 10-2032913, which is a prior document. In addition, as shown in FIGS. 2a-2b and 3a-3b of the present invention, it was confirmed that the mutant KRA16-334M70 strain produced the herbicidal active substance.

The composition may further include an auxiliary agent, and the auxiliary agent is castor oil polyoxyethylene ether, secondary alcohol ethoxylate, or polyoxyethylene lauryl ether. It may be one or more selected from the group consisting of.

In a specific example of the present invention, among 40 kinds of adjuvants, the effect of enhancing herbicidal activity by adding three kinds of adjuvants of castor oil polyoxyethylene ether, secondary alcohol ethoxylate, and polyoxyethylene lauryl ether was very good. When only the M70 culture medium was treated, the herbicidal activity was 20% in the case of indica, but when three kinds of supplements were added and treated, the herbicidal activity was increased to 100%. When treated by adding each, the herbicidal activity was enhanced to 100% (Table 4, FIG. 7). In addition, when the M-70 culture medium was diluted 2, 4, 8, 16, and 32 times, and the selected three supplements were added at a concentration of 0.1%, the herbicidal activity was greatly improved compared to the M-70 strain culture medium alone treatment group. , In particular, the secondary alcohol ethoxylate treatment showed the most obvious herbicidal activity effect (Table 6, FIG. 8).

The adjuvant may be included in an amount of 0.01 to 0.5% (v/v), preferably in an amount of 0.025 to 0.4% (v/v), more preferably in an amount of 0.025 to 0.1% (v/v), based on the total amount of the composition. v) can be included.

In a specific embodiment of the present invention, as a result of testing plant toxicity according to the concentration of the three adjuvants, castor oil polyoxyethylene ether (COG25) was not at all at pre-treatment concentrations of 0.4, 0.2, 0.1, 0.05, and 0.025%. There was no phytotoxicity, and in the case of polyoxyethylene lauryl ether (LS270), 30% phytotoxicity was expressed only at 0.4%, and there was no effect at the concentration below. In the case of secondary alcohol ethoxylate (SF90), at concentrations of 0.4, 0.2, 0.1, 0.05, and 0.025%, 90, 30, 0, 0, and 0%, respectively, it was found that at concentrations of 0.2% or more, weakness was induced.

The culture solution of the strain may be diluted 1 to 32 times, preferably 1 to 16 times, and more preferably 1 to 8 times.

In a specific embodiment of the present invention, when the mutant M-70 culture medium alone is treated with indica, the control effect in 2, 4, 8, 16, and 32-fold dilutions is insignificant at 40, 30, 20, 10, and 0%, respectively. was However, when castor oil polyoxyethylene ether (COG25) was added, the control effect of indica at the same concentration was 100, 100, 98, 80, and 70%, respectively, and when polyoxyethylene lauryl ether (LS270) was added, The control effect of was significantly increased to 100, 100, 100, 100, and 40%, respectively. In addition, when secondary alcohol ethoxylate (SF90) was added, the control effect was completely controlled at 100% at all treatment concentrations, and the herbicidal activity enhancement effect was the most excellent among the three adjuvants.

The weeds may be herbaceous weeds, broad-leaved weeds, or weeds of Dillaceae. Specifically, the flowers and weeds are sorghum ( Sorghum bicolor ), dolphin ( Echinochloa crusgalli ), indica ( Digitaria ciliaris ), American black millet ( Panicum dichotomiflorum ), wheatgrass ( Elymus tsukushiensis var. aequalis var. amurensis ), foxtail grass ( Setaria viridis ), reed ( Phragmites australis ), and silver grass ( Miscanthus sinensis ), and the broadleaf weed may be any one selected from the group consisting of Solanum nigrum , Aeschynomene indica , ( Abutilon theophrasti ), Cockle ( Xanthium strumarium ), Bindweed ( Calystegia sepium var. japonica ), Forget-me-not ( Conyza canadensis ), Shamrock ( Trifolium repens ), Wormwood ( Artemisia princeps ), Shepherd ( Capsella bursapastoris ), Amaranth ( Amaranthus tricolor ), It may be any one selected from the group consisting of Monochoria vaginalis , Potamogeton distinctus , and Bidens tripartita , and Cyperus amuricus, Cyperus serotinus , and Eleocharis kuroguwai . ), barberry ( Scirpus maritimus ) and tadpole goreng ( Scirpus juncoides var. hotarui ).

Since the substance represented by Formula 1 has weed control activity against the above-listed flowering plants and weeds, broadleaf weeds, or weeds of Dillaceae (described in Patent No. 10-2032913), the production yield of the substance represented by Formula 1 is improved. The composition containing the mutant strain KRA16-334M70 strain or a culture solution thereof has weed control activity against the above-listed flowering plants and weeds, broadleaf weeds, or weeds of Dillaceae.

The present invention provides a weed control method comprising the step of treating Streptomyces sp. mutant KRA16-334M70 strain (KCTC15096BP) or its culture medium to all or part of weeds, their seeds or their habitat. The treatment is preferably a weed foliar treatment, but is not limited thereto.

The weeds may be herbaceous weeds or broadleaf weeds. Specifically, the weed is one species selected from the group consisting of Sorghum bicolor , Dolphin ( Echinochloa crus-galli ), Indica ( Digitaria sanguinalis ), Agropyron smithii , and American dog millet ( Panicum dichotomiflorum ) as flowers and weeds. It may be two or more species, and the weeds may be one species selected from the group consisting of Solanum nigrum , Aeschynomene indica , Abutilon avicennae , Xanthium strumarium , and Bindweed ( Calystegia japonica ) as broadleaf weeds, or There may be two or more.

Example

Hereinafter, examples will be described in detail to explain the present invention in detail. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Materials and Methods

1. Mutagenesis by UV light

Streptomyces drozdowicchii KRA16-334 strain (accession number: KCTC13357BP) spore suspension (10 ⁷ colony / ml) was diluted (10 ^-3 ) using sterile distilled water, and then spread on an agar medium for culturing actinomycetes and under cancer conditions After irradiating with UV (254 nm) for a certain period of time, the number of colonies was investigated for each time in order to explore the conditions for a colony formation rate of 1% (FIG. 1).

In the case of the wild-type KRA16-334 strain, the average number of colonies in the untreated control group was 1250, and the average number of mutant colonies formed by UV irradiation for 45 seconds at a height of 17 cm for the wild-type strain was 13.8 on average, which was 1.1% higher than that of the untreated control group. Colonies were formed (Table 1). Subsequently, mutagenesis by ultraviolet light was performed using the conditions of FIG. 1 and an ultraviolet irradiation time of 45 minutes.

In addition, each colony selected through strain improvement was inoculated into a liquid culture medium (M3; Soytone 1%, Starch 2%, Glucose 1%, CaCO ₃ 0.3%, FeSO ₄ 0.02% /L), respectively, at 27℃ and 150℃. After culturing with shaking for 7 days at rpm, herbicidal activity evaluation and HPLC quantitative analysis were performed using the culture filtrate. In order to evaluate the herbicidal activity of each culture medium, the culture filtrate was diluted 50 times and sprayed on indian plants, and after 7 days, the activity was evaluated through a decantation survey. After analysis, the most active mutants were finally selected.

2. Chemical mutagenesis

1 hour or 3 hours by adding a certain amount of NTG (N-methy-N-nitro-N-nitrosoguanidine, MNNG) solution to the spore suspension (2x10 ⁵ spore/ml) of the mutant strain first selected through mutation by ultraviolet light After culturing, conditions such as incubation time, treatment concentration of NTG, etc. were investigated, and conditions such as 50-60% bacterial death were investigated and used for selection of mutant strains. In addition, sodium nitrate was added to strains selected through mutagenesis by ultraviolet light, and chemical mutagenesis was induced in the same manner.

3. Measurement of effective herbicidal active substance content of improved strains through HPLC analysis

Analysis of the content of the effective herbicidal active substances produced by the KRA16-334M70 strain in the culture medium was carried out at a flow rate of 0.8 ml/min under each solvent condition using an atlantisT3 column (C18 column; 4.6X250mm) from Waters, with active substances at 210 nm and 230 nm. The peaks of were identified and the area of each peak was calculated.

The developing solvent for quantitative analysis of the active material 334-W4 was aq. 30% MeOH was used, and in the case of 334-M100, the active material was separated using 55-100% acetonitrile + 0.02% TFA. For each active substance, a quantitative graph was prepared using the purified compound, and then the content of the substance in the culture solution obtained through mutagenesis and establishment of culture conditions was confirmed through HPLC analysis. The peak area of retention time) was estimated using a formalized quantitative graph (Figs. 2a-2b, 3a-3b).

4. Adjuvant of composition for controlling weeds

(1) Concentration setting of KRA16-334M70 strain culture solution for selection of adjuvants

In a polystyrene pot with a surface area of 38.5 cm ² , 7 to 10 seeds of Inari and black nightshade stored in a low temperature were sown and grown for 13 days in a greenhouse condition at 28 ± 5 ° C (light / dark = 14 / 10 hrs). After incubation, the M70 strain culture medium stored in the refrigerator was left at room temperature for a certain period of time, and the treatment concentration was prepared to be 50, 25, 12.5, 6.2, 3.2, 1.6, 0 (%), on the other hand, Tween-20 at the same concentration It was prepared to be 0.1%.

Foliage treatment was carried out in the amount of 5 ml/pot of the preparation solution for each concentration in the pots in which indica and black nightshade were sown, and managed under the same greenhouse conditions. After 7 days of treatment, the degree of herbicidal activity was visually inspected (0; no drug effect, 100; complete control). Based on the results of the telescopic survey, the 70% inhibitory concentration (I ₇₀ value) was calculated using a regression linear equation, and the herbicidal activity between the M70 strain culture medium and the treatment group added with Tween-20 was compared.

(2) Selection of supplements

In a polystyrene pot with a surface area of 38.5 cm ² , 7 to 10 seeds of Inari and black nightshade stored in a low temperature were sown and grown for 12 days in a greenhouse condition at 30 ± 5 ° C (light / dark = 14 / 10 hrs). After culturing, the M70 strain culture medium stored in the refrigerator was left at room temperature for a certain period of time, and the treatment concentration was prepared to be 10%. v) Each was prepared by adding to a concentration. The above 40 kinds of supplements were received from Mokwoo Research Institute and used. Foliage treatment was performed at 5 ml/pot of each treatment preparation solution and managed in the same greenhouse conditions, and then, after 5 and 10 days of treatment, the degree of herbicidal activity due to each adjuvant and the addition of each adjuvant was visually examined (0; no drug effect, 100; complete control).

(3) Herbicidal activity of M70 strain culture broth by selected adjuvants

In a polystyrene pot with a surface area of 38.5 cm ² 7 to 10 grains of indica seeds stored in a low temperature were sown and grown for 11 days in a greenhouse condition at 30 ± 5 ° C (light / dark = 14 / 10 hrs). To evaluate the weakness of three types of supplements, castor oil polyoxyethylene ether (COG25), secondary alcohol ethoxylate (SF90), and polyoxyethylene lauryl ether (LS270), which were selected for the first time, each adjuvant was 0.4, 0.2, 0.1, 0.05, and 0.025% were prepared and treated.

In addition, in order to evaluate whether herbicidal activity is enhanced by the addition of the selected three adjuvants, the M70 strain culture medium was prepared in 2, 4, 8, 16, and 32-fold dilutions, and then treated with or without the addition of 0.1% of the adjuvant. At this time, all treatment groups were treated with foliar treatment in an amount of 5 ml / pot and managed in the same greenhouse conditions, and after 4 days of treatment, the degree of damage or control effect was visually inspected (0; no drug effect, 100; complete control). After 7 days of one treatment, systemic treatment was performed with the same concentration and amount of treatment, and all treatments were performed in 3 repetitions. Indica, which is stored in a low-temperature dry state, was sown in a square plastic pot with a surface area of 350 cm ² , and then transplanted into a pot with a surface area of 95 cm ² when cotyledons appeared above ground. The degree of growth was around 4-5 leaf stages.

result

1. Strain improvement using UV mutation

As a result of evaluating the herbicidal activity of 182 mutant strain cultures selected through strain improvement by ultraviolet irradiation, it was confirmed that the herbicidal activity of 18 mutant strains was increased compared to the wild type. Some of the results of the multi-objective investigation after treatment of the 18 mutant strains are shown in FIG. 4.

In 11 of the 18 mutant strains, the content of the active substance 334-W4 was significantly increased compared to the wild type, and in 7 mutant strains, it was confirmed as a result of quantitative analysis that the content of 334-100M was increased compared to the wild type (FIG. 5 ).

Among the mutant strains with increased yield of active substance 334-W4, strain M70 with the highest yield was selected, and after confirming that the active substance was stably produced through recovery prevention, it was stored using a glycerol (30%) storage solution. The strain was created and stored at −70° C., and then used as an inoculum in experiments.

Mutant strain M70 showed a yield increase of about 4 times that of the wild type in the case of active substance 334-W4 (FIG. 5), and showed 10 times or more herbicidal activity compared to the control group when evaluating the herbicidal activity according to the lunar survey (FIG. 4). Therefore, the mutant strain M70 exhibited the best herbicidal activity and increased active substance content, and was selected as the mutant strain of the present invention.

2. Adjuvant of composition for controlling weeds

(1) Concentration setting of KRA16-334M70 strain culture solution for selection of adjuvants

In the case of treating only the M-70 strain culture solution, it was attached to the plant surface in the form of droplets due to surface tension, resulting in a very poor electrodeposition effect. It was found to spread evenly on the surface of the plant and to have good spreading power.

Referring to Table 3, after 7 days of drug treatment, in the case of black nightshade, when only the M70 strain culture was treated, the herbicidal activities at 50, 25, 12.5, 6.2, 3.2, and 1.6 (%) were 94.3, 70.0, 56.7, 33.3, 10.0 and 0%, and the herbicidal activity when Tween-20 was added was 99.3, 95.0, 93.3, 76.7, 50.0, and 36.7%, respectively, showing a clear effect of enhancing herbicidal activity. On the other hand, in the case of Indica, when only the M70 strain culture was treated, the herbicidal activities at the same concentrations were 36.7, 20.0, 13.3, 5.0, 0, and 0%, respectively, and when Tween-20 was added, the herbicidal activities were 99.3, 97.0, and 70.0, respectively. , 56.7, 50.0, and 33.3%, respectively, the herbicidal activity was greatly improved.

¹⁾ Herbicidal activity was evaluated by multi-objective survey (0%: no effect, 100%: complete death) after 7 days of foliage treatment.

* BF: Broth filtrate of strain M70, T: Tween-20 ^ⓡ (0.1%)

Figures 6a and 6b show the calculation of 70% inhibitory concentration using a regression linear equation based on the results of Table 3 [Figure 6a: M70 strain culture filtrate alone, Figure 6b: M70 strain culture filtrate + Tween-20 ( 0.1%)]. Referring to FIG. 6a, when only the M70 strain culture was treated, 70% inhibitory concentration could not be calculated in the case of indica, and the 70% inhibitory concentration of black nightshade was 28.50%. Referring to Figure 6b, when Tween-20 was added to the M70 strain culture medium, the 70% inhibitory concentration of Indica was 13.21%, and the 70% inhibitory concentration of black nightshade was 7.02%. Therefore, the 70% inhibitory concentration by the addition of Tween-20 could not be compared in the case of indica, but it was confirmed that it was increased by about 4.1 times in the case of black nightshade.

(2) Selection of supplements

The herbicidal activity of the mutant strain M70 culture medium was evaluated by the addition of 40 kinds of adjuvants, and the effect of enhancing herbicidal activity was more evident in the flower bud and weed indica than in the broadleaf weed Camellia. In particular, COG25 [Castor oil polyoxyethylene ether], SF90 [Secondary alcohol ethoxylate], LS270 [Polyoxyethylene lauryl ether] The effect of enhancing herbicidal activity was very good. When only the M70 culture medium was treated, the herbicidal activity was 20% in the case of indica, but when three kinds of supplements were added and treated, the herbicidal activity was increased to 100%. When treated by adding each, the herbicidal activity was enhanced to 100% (Table 4, FIG. 7). Therefore, castor oil polyoxyethylene ether (COG25), secondary alcohol ethoxylate (SF90), and polyoxyethylene lauryl ether (LS270) were selected as adjuvants for enhancing the herbicidal activity of the mutant strain M70 culture medium.

On the other hand, in the case of only adjuvant treatment, observation after 5 days of treatment showed weakness such as burn-down at the tip of the leaf or water soaked in the center of the leaf in some species of the adjuvant, but 10 days after treatment Afterwards, all of them recovered and were not observed with the naked eye, so the weakness of the supplement itself was judged to be negligible.

¹⁾ Plant toxicity and herbicidal activity were evaluated by multi-objective survey (0%: no effect, 100%: complete death) 10 days after foliar treatment.

(3) Herbicidal activity of M70 strain culture broth by selected adjuvants

Of the 40 adjuvants, three adjuvants with excellent herbicidal activity enhancement effects were confirmed: castor oil polyoxyethylene ether, secondary alcohol ethoxylate, and polyoxyethylene lauryl ether, and the degree of weakness against indica. Castor oil polyoxyethylene Ether (COG25) showed no weakness at all treatment concentrations of 0.4, 0.2, 0.1, 0.05, and 0.025%, and polyoxyethylene lauryl ether (LS270) showed 30% weakness only at 0.4%. had no effect on In the case of secondary alcohol ethoxylate (SF90), at concentrations of 0.4, 0.2, 0.1, 0.05, and 0.025%, respectively, 90, 30, 0, 0, and 0% were found to induce weakness at concentrations of 0.2% or more (Table 5) .

However, since the mutant strain M-70 exhibits non-selective herbicidal activity, it will not be treated on crops unless necessary, so the deterioration of the supplement itself is not a big problem, and at concentrations of 0.1% or less Since no harm itself occurs, it was judged that it would be okay to select as an adjuvant for enhancing herbicidal activity.

¹⁾ Plant toxicity was evaluated by multi-objective survey (0%: no effect, 100%: complete death) 4 days after foliar treatment.

When the M-70 culture medium was diluted 2, 4, 8, 16, and 32 times, and the selected three supplements were added at a concentration of 0.1%, the herbicidal activity was greatly improved compared to the M-70 strain culture medium alone treatment group. In particular, the secondary alcohol ethoxylate (SF90) treatment showed the most distinct herbicidal activity. When the mutant strain M-70 culture medium alone was treated on indica, the control effect at 2, 4, 8, 16, and 32-fold dilutions was insignificant at 40, 30, 20, 10, and 0% after 4 days of treatment, respectively. It is believed that this is because when the mutant strain M-70 culture medium was treated alone, the effect of adhesion and spread to the plant surface was lowered due to the morphological characteristics of flowers and weeds and the surface tension of the culture medium. However, when castor oil polyoxyethylene ether (COG25) was added, the control effect of indica at the same concentration was 100, 100, 98, 80, and 70%, respectively, and when polyoxyethylene lauryl ether (LS270) was added, The control effect of was significantly increased to 100, 100, 100, 100, and 40%, respectively. In addition, when secondary alcohol ethoxylate (SF90) was added, the control effect was completely controlled at 100% at all treatment concentrations, and the herbicidal activity enhancing effect was the most excellent among the three adjuvants (Table 6, FIG. 8).

¹⁾ Herbicidal activity was evaluated by multi-tube survey (0%: no effect, 100%: complete death) 4 days after foliar treatment.

Therefore, all three selected adjuvants effectively enhance the herbicidal activity of strain M-70, and among them, secondary alcohol ethoxylate (SF90) was evaluated to be the most effective.

So far, specific examples of the composition for controlling weeds including the Streptomyces genus mutant strain KRA16-334M70 or its culture medium of the present invention have been described, but various modifications are possible within the limits that do not deviate from the scope of the present invention is self-explanatory.

Therefore, the scope of the present invention should not be limited to the described embodiments and should not be defined, and should be defined by not only the claims to be described later, but also those equivalent to these claims.

That is, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive, and the scope of the present invention is indicated by the claims to be described later rather than the detailed description, and the meaning and scope of the claims and All changes or modified forms derived from the equivalent concept should be construed as being included in the scope of the present invention.

Claims (12)

In the composition for controlling weeds containing the culture solution of the Streptomyces sp. mutant KRA16-334M70 strain (KCTC15096BP) as an active ingredient,
The composition further comprises, as an adjuvant, castor oil polyoxyethylene ether, secondary alcohol ethoxylate or polyoxyethylene lauryl ether,
Characterized in that the culture solution of the strain is diluted 1 to 32 times,
A composition for controlling weeds.
According to claim 1,
The strain is
Characterized in that the yield of herbicidal active substances is increased compared to Streptomyces sp. strain KRA16-334 (KCTC13357BP),
A composition for controlling weeds.
According to claim 2,
The composition
Characterized in that it comprises a substance represented by Formula 1 as a herbicidal active substance,
A composition for controlling weeds.
[Formula 1]

delete delete According to claim 1,
The adjuvant is characterized in that it is included in 0.01 to 0.5% (v / v) with respect to the entire composition,
A composition for controlling weeds.
delete According to claim 1,
The composition
Characterized in that it has control activity against flowers and weeds or broadleaf weeds,
A composition for controlling weeds.
According to claim 1,
The composition
One or two or more plants selected from the group consisting of Sorghum bicolor , Echinochloa crus-gall i, Indica ( Digitaria sanguinalis ), Agropyron smithii , and Panicum dichotomiflorum as flowers and weeds Characterized in that it has weed control activity for,
A composition for controlling weeds.
According to claim 1,
The composition
As broadleaf weeds, the weed control activity against one or more plants selected from the group consisting of Solanum nigrum , Aeschynomene indica , Abutilon avicennae , Xanthium strumarium and Calystegia japonica characterized by having
A composition for controlling weeds.
In a weed control method comprising the step of treating all or part of weeds, their seeds or their habitat with a composition containing the culture medium of the Streptomyces sp. mutant KRA16-334M70 strain (KCTC15096BP) as an active ingredient ,
The composition further comprises, as an adjuvant, castor oil polyoxyethylene ether, secondary alcohol ethoxylate or polyoxyethylene lauryl ether,
Characterized in that the culture solution of the strain is diluted 1 to 32 times,
weed control methods.
According to claim 11,
the weed
Characterized in that it is a flower and a weed or a broadleaf weed,
weed control methods.

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