KR100477890B1 - A new Streptomyces sp. 8E12 and its use as a herbicidal agent - Google Patents

Abstract

The present invention relates to Mycobacterium strain Streptomyces sp. 8E12 and herbicides using the same, and more particularly, to Mycobacterium strain Streptomyces sp. 8E12, culture medium, extracts, and amino acids isolated therefrom. Bio-pesticides, because they contain glycoside antibiotic methoxyhygromycin or its derivative epi-methoxyhygromycin as an active ingredient and show strong herbicidal activity against domestic rice fields and field weeds It relates to a new herbicide which can be used as.

Description

Genus 12 of the genus New strain Streptomyces and herbicides using the same {A new Streptomyces sp. 8E12 and its use as a herbicidal agent}

The present invention relates to Mycobacterium strain Streptomyces sp. 8E12 and herbicides using the same, and more particularly, to Mycobacterium strain Streptomyces sp. 8E12, culture medium, extracts, and amino acids isolated therefrom. Bio-pesticides, because they contain glycoside antibiotic methoxyhygromycin or its derivative epi-methoxyhygromycin as an active ingredient and show strong herbicidal activity against domestic rice fields and field weeds It relates to a new herbicide which can be used as.

Weed species in agricultural ecosystems are very numerous and the damage is enormous. Weeds are an important limiting factor in reducing crop productivity by competing with crops for the moisture, nutrients, and sunlight that are essential for crop growth. Although various types of organic synthetic herbicides are currently used for weed control, the production loss of crops by weeds is still high, which is due to the continuous use of herbicides, causing ecological changes in weed colonies, herbicide resistance, and currently used herbicides. This is because new weeds are difficult to control. Until now, the development of new microbial herbicides with low toxicity and environment friendliness has been required due to the serious pollution and human toxicity problems caused by the large use of organic synthetic herbicides for weed control. As a result, more than 100 kinds of microorganisms (mostly fungi) have been found to be effective in controlling weeds in the past 30 years. Currently, about 10 kinds of microbial herbicides are registered and used. In recent years, the control of dominant weed species by traditional methods has reached its limit, and the necessity of using biological control methods in parallel with the seedling method has emerged. In terms of preservation of diversity, it is a very appropriate control method.

As reported in connection with the development of microbial herbicides using the current pathogenic microorganism strain Sickle Pod (sicklepod) control as Alterna Ria Cassia (Alternaria cassiae), Spud cyano is (spurred anoda) as for the control Alterna Ria MACROSS Fora (Alternaria for macrospora ), for the control of Italian thistle, Alternaria sp., and for the control of prickly sida, most of the fungi, such as the colletotrichum malvarium .

On the other hand, in Korea, a microbial herbicide has been patented to control the alligator , one of the weeds using Epicoccosorus strain, and Exero Hill Room to control the most problematic blood among the weeds. A study is being conducted to isolate and use a monosera strain ( Exserohilum monoceras ). In addition, the development of biologics using Plectosporium tabacinum for control of peel and snare has been attempted. In 1991, ill lesions showing symptoms of late blight were collected to isolate Phytophthora erythroseptica late blight strains, and the spores of the late blight were re-inoculated to observe the incidence and suggest potential as biologics. I've done it. Recently, the Korea Research Institute of Bioscience and Biotechnology has been able to effectively control several types of field weeds, such as shamrock ( Trifolium repens ), Solanum nigrum , Aeschynomene indica , Xanthium strimarium , and Calystegia japonica . It shows the possibility of using as a microbial herbicide for field weed control by selecting oxalicum strain, developing it as a microbial herbicide for grassland and registering it as a patent, and using the myrothecium roridum strain. We have developed and registered a patent for biologics for weed control.

Such microbial herbicides have been accepted as a natural way to control conventional herbicides. Recently, a lot of researches have been applied to the application of phytotoxins derived from microorganisms for weed control. To date, herbicide microbial metabolites include cycloheximide, herbimycin, herbicidin, anisomycin, and posalacin. 30 Several metabolites have been reported. Among them, bialaphos produced by Actinomycete Streptomyces hygroscopicus is the most commercially successful microbial herbicide. In addition, Mycogen has studied the development of herbicides using Streptomyces cassia strain. However, no studies have been attempted for biological weed control using actinomycetes itself and actinomycetes-derived herbicides in Korea.

Therefore, the present inventors were studying and developing a method for controlling the major domestic weed species by biological methods using soil-derived actinomycetes, and isolated a new actinomycete strain showing a high herbicidal effect on domestic weed species, and identified the actinomycetes Strepto It was confirmed that the genus Streptomyces sp. Weed control effect of the weeds using methoxyhygromycin or epi-methoxyhygromycin isolated from the strain itself (spores, etc.), fungi culture and major herbicidal active substances. The present invention was completed by confirming.

Accordingly, the present invention provides a novel strain of Streptomyces genus 8E12 (KCTC 10336BP), a culture medium, an extract, and a methoxyhygromycin or epi-methoxyhygromycin isolated from the weed species. Its purpose is to provide used herbicides.

The present invention is characterized by the genus Streptomyces genus 8E12 (KCTC 10336BP).

In addition, the Streptomyces genus 8E12 (KCTC 10336BP) strain itself, the culture medium, extract, and the active substance methoxyhygromycin (epi-methoxyhygromycin) or epi-methoxyhygromycin (pi-methoxyhygromycin) isolated from this herbicide It is confirmed that there is activity and available features as biopesticides are another feature.

Referring to the present invention in more detail as follows.

The present inventors collected many soil samples from all over the country to search for new microorganisms for controlling plant diseases, and isolated strains showing selective herbicidal activity from the collected soil samples using Bennett's agar medium. The isolated actinomycetes were cultured in liquid, and the best strains were selected by performing in vitro petri plate on seedling and seed germination inhibition and in vivo potting experiment. In addition, methoxyhygromycin and epi-methoxyhygromycin, which are aminoglycoside herbicidal active substances, were separated from the pure water, their chemical structures were investigated, and their in vivo herbicidal activity was confirmed.

Referring to the identification, culture, activity assay and separation of the active substance of the strain as follows.

end. Isolation, Culture and Identification of Strains

1) Isolation and Identification of Bacteria

According to the International Streptomycets Project (ISP) regulations, the cells were incubated at 28 ° C. for 14 days and their morphological characteristics were examined under an optical microscope and an electron microscope. (rectiflexibiles) type, the number of chain spores was 5-10 or more than 10, the surface of the spores were smooth, the spores were cylindrical and the size was 0.6 ~ 0.7 × 1.0 ~ 1.2.

2) Cultural Characteristics

After culturing at 28 ° C. for 14 days in various ISP medium conditions, the culture physiological characteristics of the strain were examined, and the results are shown in Table 1 below. The growth of this strain showed relatively good growth in yeast, malt agar and tyrosine agar medium and well formed water-soluble pigments. In other media, mycelial growth was poor as well as mycelial growth.

Type of badge Spore color Mycelia Substrate hyphae Water-soluble pigment Growth color Growth color Yeast-malt agar medium (ISP 2) White usually White poorness Yellow chestnut Yellow chestnut Auto Mill Badge (ISP 3) White Medium (poor) White  usually White Yellow chestnut Inorganic Salt-Starch Agar Medium (ISP 4) White-grey Dwarf White-grey usually Yellow brown Yellow chestnut Glycerol-asparagine Agar Medium (ISP 5) White Very dwarf White usually Yellow chestnut Yellow chestnut Tyrosine Agar Badge (ISP 7) White-grey usually White-grey usually Yellow chestnut Melanoid

3) Physiological characteristics

In order to investigate the physiological characteristics of the strains, starch hydrolysis, gelatin liquefaction, skim milk cohesion, melanoid formation ability, phosphate hydrolysis ability, saline resistance after incubation at 28 ° C. for 14 days using Schilling-Gertib agar medium. The degree was investigated and shown in Table 2 below.

Survey item Characteristics Melanin Pigment Production positivity Gelatin Liquefaction voice Water Soluble Pigment voice Starch hydrolysis voice  Schememilk coagulation voice Flameproof degree 3% less than

4) Carbon Source Availability

In order to investigate the carbon source availability of the strain, using the Schilling-Gurtrib agar medium was incubated at 28 ℃ for 14 days and the carbon source availability was investigated. The results are shown in Table 3 below. The strain used well D-glucose, L-arabinose, myo-inositol, D-mannitol, raffinose, galactose, and D-fructose among 12 carbon sources disclosed, but did not use D-gyros well. Rhamnose, salicylic acid, sucrose, cellulose and the like were not used at all.

Party Usability D-glucose positivity L-arabinose positivity D-fructose positivity Galactose positivity Myo-inositol voice D-mannitol positivity Raffinose positivity Rhamnose voice Sucrose voice D-xylose weak Cellulose voice Salincin voice

5) Cell composition

The bacterial cell wall components of the strain were investigated according to Becker et al. [Becker et al., Applied Microbiology 12: 421-423, 1964], and the amino acid hydrolysis component diamino-pimellic acid of the strain was LL. Brother.

As described above, the strain was identified as a strain belonging to the genus Streptomyces according to the results of the morphological, culture, physiological characteristics or sugar availability, and cell composition of the present strain, and the strain was identified as Streptomyces sp. 8E12) and was deposited with the Korea Biotechnology Research Institute Gene Bank (KCTC) on September 11, 2002 and was given accession number KCTC 10336BP.

2. Cultivation of Strains

The strain is cultured in a medium containing nutrients that can be used by ordinary microorganisms. Glucose + soluble starch (GSS) medium was used as a fermentation medium, coarse soy flour, yeast extract, and gravy were used as a nitrogen source, and dipotassium hydrogenphosphate was added, and calci was adjusted for pH control. Incubate with calcium carbonate. As a culture method, shaking culture was carried out in a fermenter (jar fermentor) for 5 liters under aerobic conditions, and the culture temperature was 28 ℃. The inoculum to be inoculated in the fermentation medium is precultured for 3 days in the Erlenmeyer flask and inoculated at 3% level in each fermenter. Antifoaming agent added neoin (neorin), and the aeration rate of 1 vvm, 280 rpm when incubated at a strain density of 10 ⁹ or more per 1 ml in the Streptomyces genus The optimal medium was selected for 8E12 culture.

Meanwhile, Bennett's broth, which is commercially available, contains components selected from liquid culture for solid mass cultivation of Streptomyces spp. 8E12 strain, which is adsorbed in perlite and sterilized in a culture bottle, followed by sterilization of Streptomyces spp. 8E12 As a result of inoculating strains, 7 days after the inoculation, the density of 10 ⁹ per g of the spores was investigated in the adsorbed pearlite, and the water was easily absorbed.

3. Herbicide Activity Assay

For investigation of the culture conditions, a part of the culture was taken every 24 hours to assay the herbicidal activity of the culture.

In vitro and in vivo activity assays were performed on 14 weed species using small pots for isolated strains.

4. Identification of active substance separation structure

In order to separate and purify the active substance, the culture supernatant obtained by centrifugation of the culture solution cultured on the fermenter is removed. First, the culture supernatant was adsorbed by passing through an activated carbon column, washed with acetone, eluted, and concentrated under reduced pressure. The concentrated solution was then extracted three times with butanol, and the active fraction butanol layer was concentrated under reduced pressure and developed on a silica gel column containing a mixture of chloroform and methanol as a solvent to obtain an active fraction, which was then concentrated under reduced pressure and subjected to column chromatography using methanol as a solvent. Isolate and analyze on HPLC to obtain pure herbicidal actives. The molecular weight (Mass), nuclear magnetic resonance (NMR), ultraviolet (UV) analysis of the isolated active material, methoxyhygromycin and epi-methoxyhygromycin, aminoglycodisde antibiotics The identity was verified and the separation ratio was separated at a ratio of approximately 3: 1.

Hereinafter, weed control using the mycobacterial Streptomyces sp. 8E12 itself, herbicidal active material methoxy hygromycin and its derivatives will be described in detail by the following examples. The present invention is not limited to these. Of course, the present invention is not limited only to the examples described below, and indicates that activity can be exhibited even if the weed species to be controlled, the type of the medium, the culture conditions, etc. are greatly changed.

Example 1: Liquid Culture of Genus 8E12 (KCTC 10336BP) Genus Streptomyces

Cultivation of this strain as seed and production media was cultivated in a medium containing nutrients that can be used by ordinary microorganisms. Glucose 20%, Soluble starch 10%, Rough soy flour 25%, Juicy 1%, Yeast extract 4%, NaCl 2%, K ₂ HPO ₄ 0.25%, CaCO ₃ 2% after adjusting to pH 7.2 for use It was. 50 ml each of two 500 ml baffled flasks were sterilized for 20 minutes at 121 ° C., inoculated with agar pieces of the strain and shaken for 2 days at 28 ° C. and 150 rpm. It was used as species culture of the incubator. A 5 liter fermenter containing 1000 ml sterilized at 121 ° C. for 20 minutes was inoculated with 3% of the seed culture solution, followed by shaking culture at 28 ° C. for 7 days, and 0.2% of neoin was added as an antifoaming agent. vvm, incubated at a speed of 280 rpm.

Example 2: Solid culture of 8E12 (KCTC 10336BP) genus New strain Streptomyces

For solid mass cultivation of Streptomyces sp. 8E12 strains, Bennett's broth, a commercially available ingredient containing glucose, yeast extract, peptone, beef broth, and vitamins, was adsorbed on perlite to 800 Sterilized in cc culture bottles, and stored in Streptomyces spp. As a result of inoculation with 8E12 strain, 7 days after the inoculation, perlite of adsorbed pearlite produced high density of 10 ⁹ per g, the pearlite adsorption method could be used as a useful method for culture of actinomycetes spores. Can be.

Example 3: Separation of Herbicides

The herbicidally active substance was purified purely using the culture solution obtained in the above-described embodiment. For separation and purification, the culture supernatant was removed by centrifugation and the culture supernatant was used. First, the culture supernatant was passed through an activated carbon column, adsorbed, washed with 30% acetone and concentrated under reduced pressure by eluting the active fraction with 80% acetone. The concentrate was extracted three times with butanol, and the active fraction butanol layer was concentrated under reduced pressure, and then developed on a silica gel column (300 cc) using chloroform: methanol (4: 1, v / v) as a solvent to obtain an active fraction and concentrated under reduced pressure. Thereafter, the mixture was separated on Sephadex LH-20 and Sephadex G-10 using methanol as a solvent, and analyzed on HPLC to obtain 40 mg of pure herbicide. In this process, the amount of methoxyhygromycin and epi-methoxyhygromycin separated was approximately 3: 1.

1) UV spectrum

UV spectra were measured by dissolving the sample in MeOH. In the silica gel thin layer chromatography plate, when chloroform: methanol (4: 1, v / v) was used as the developing solvent, Rf value 0.22, butanol: methanol: water (4: 1: 1, v / v) was used as the developing solvent. Rf value 0.70 was shown. The characteristics of the herbicidal active substance isolated from 8E12 bacterial cultures of Streptomyces were investigated. As a water-soluble substance, it was very soluble in water and soluble in methanol, but insoluble in other organic solvents, ethyl acetate, chloroform and benzene. The UV-absorbing properties of the herbicidal active material produced by 8E12 strain of Streptomyces were 214, 270 and 300 nm in neutral, and no shift in acid but 250, 280 in alkaline. 322 nm.

2) Mass (SI-MS) Spectrum

As a result of analyzing the selective ion mass spectroscopy (SI-MS) spectra to investigate the molecular weight, as shown in FIG. 2, the (M + H) ⁺ ion peak was 514 and the actual molecular weight was 513. The conventional methoxyhygromycin It was confirmed that the same substance as.

3) Hydrogen- and carbon-nuclear magnetic resonance ( ¹ H- and ¹³ C-NMR) spectra

¹ H-NMR and ¹³ C-NMR spectra were examined for structure identification. TMS was used as a standard and CD ₃ OD was used as a solvent. As a result of analyzing the ¹ H-NMR and ¹³ C-NMR spectra as shown in Figures 3 and 4, the molecular structure is represented by the following Chemical Formula 1 as C ₂₃ H ₃₁ NO ₁₂ , the conventional Yoshida et al. (1990) and Chida et al. (1991) It appears to match the report.

Physicochemical Properties of Methoxyhygromycin Isolated from New Strains Constellation White powder Molecular Weight (SI-MS, m / z) 514 (M + H) ⁺ UV maximum absorption wavelength (methanol) nm 214 270 300 (s) UV maximum absorption wavelength (MeOH-HCl) nm 214 270 300 (s) UV maximum absorption wavelength (MeOH-NaOH) nm 250 280 (s) 322 Solubility Water soluble in methanol; Water-insoluble in ethyl acetate, chloroform, benzene Thin layer chromatography (silica gel) 0.22 (chloroform: methanol, 4: 1, v / v)

As a result, the herbicidal active substance was identified as methoxyhygromycin and epi-methoxyhygromycin similar to hygromycin-A. Until now, the antibacterial activity of methoxyhygromycin has been reported. However, this study is the first time for herbicidal activity.

Example 4: in vitro  Activity test

The primary in vitro herbicidal activity of the isolates was carried out by inhibiting germination of seeds of barley and blood. Seed germination inhibition method is a method for seeding germination and seedling height after inoculating barley and seed on a wet plate, inoculated with the culture medium and extract methoxy hygromycin, and for 3 to 4 days Seed germination in advance and direct treatment on seedlings. That is, three sheets of filter paper are immersed in sterilized water and placed on a plate, and then the culture medium and extracted methoxy hygromycin are diluted to several times in the stock solution and treated at a constant concentration in germinated seedlings such as barley and blood and germinated seedlings. It is a method of denture in a humidified incubator.

As a result, it was found that the genus Streptomyces 8E12 bacteria cultures and cultures extract that high selective herbicidal activity in indica and P were subjected to a herbicidal activity test in vivo on the pot on the basis of this information.

Example 5: Under Greenhouse Conditions in vivo  Pot activity experiment

In order to select strains with pathogenic and herbicidal activity in the weeds, the primary screening is the target weeds (sorghum, dolpi, wheat, barley, American larvae, crows, larvae, tabby, lizards, buckthorn, bark, dolpi, tusks and shamrocks). Assays were treated by directly treating the culture medium or spore suspension with leaves on the leaves. Plants for in vivo herbicidal activity assay should be mixed with an appropriate amount of fertilizer in unsterilized sandy soil, and then placed in test pots (150, 350 cm ² ) to make sowing spheres, sowing weeds or crop seeds and covering them with fine soil. After growing in a greenhouse, the culture solution to be treated and the methoxy hygromycin solution (0.2% of Tween 20 solution added in a small amount) were prepared, treated with a hand sprayer, and treated with leaves.

The culture solution was diluted and diluted several times in the stock solution, and the culture extract was treated with a concentration of 0.1 to 16 mg of the effective concentration of each active substance per pot. Investigation was performed by physiological observations. Treatment was divided into pre-emergence and post-emergence to examine herbicidal activity. In the case of zero control and 100% complete control, the degree of herbicidal activity was evaluated to have a grade of 70% or higher. The first species was regarded as a controllable first species. The results are shown in the following Table 5 and FIG.

Herbicidal Effects of Methoxyhygromycin cotyledon Weed species Compound (kg / ha) 4 2 One 0.5 0.25 A monocot Sorghum bicolor (sorghum) 100 ^* 90 80 70 45 Echinochloa crus-galli (blood) 100 100 100 90 90 Agropyron smithii (wheat wheat) 65 50 40 0 0 Digitaria sanguinalis (cement) 100 100 100 80 90 Panicum dichotomiflorum (United States of America) 100 100 60 10 20 Dicotyledonous Solanum nigrum 90 50 50 0 0 Aeschynomene indica 90 90 90 80 50 Abutilon avicennae ( buzzard ) 50 40 60 20 0 Xanthium strumarium 30 20 0 0 0 Calystegia japonica ( buckthorn ) 65 60 20 0 0 ^{* The} herbicidal effect was evaluated after 14 days of treatment, 0: no effect, 10-30: fine effect, 40-60: moderate effect, 70-90: strong effect, 100: full herbicidal effect

In addition, as shown in FIG. 5, methoxyhygromycin exhibited selective herbicidal activity against barley, blood, and the like, and in particular, exhibits a feature of causing albino symptom, and thus has a pigment inhibition activity. It became.

As a result of shaking culture, it was confirmed that the production of the active substance reached the highest by incubation for 90 hours, and that the strain could be used as a biological agent with the isolated herbicide active substance.

Example 6 Herbicide Effect of Microbial Herbicide

In Example 2, 8E12 strains of the genus Streptomyces were solid-cultured to make a microbial herbicide at a concentration level of 1 × 10 ⁷ spores / g, suspended in sterile water, treated to weed species including barley, and the herbicidal activity was observed. . The herbicidal activity of the strains was evaluated by similar criteria as in Example 5 above. The results are shown in Table 6 below.

In vivo Herbicide Activity of Streptomyces 8E12 Formulation Scientific name Common name Control effect Abutilon avicennae Velvetleaf + Aeschynomene indica Indian jointvetch +++ Agropyron smithii Cheatgrass +- Calystegia japonica Bindweed +- Digitaria sanguinalis Large crabgrass +++ Echinochloa crus-galli Barnyardgrass +++ Oryzae sativa Rice +- Panicum dichotomiflorum Fall panicum + Sagittaria pygmaea Arrow head + Solanum nigrum Black nightshade +- Sorghum bicolor Sorghum ++ Xanthium strumarium Cocklebur +-  * Control price results after 4 weeks of inoculation +: <30%, +-: <50%, ++: <70%, +++: <90%

As shown in Table 6, the new strain shows herbicidal activity against various field weed species and some non-weed varieties, particularly resistant to field weed species such as barley, silkworm grass, and American cultivated fields and non-weed species such as blood It showed herbicidal effect.

As described in detail above, the present invention isolates excellent strains showing selective herbicidal activity against weed species such as barley, blood, etc., and analyzes the morphological and biochemical characteristics of the strains, and thus, Streptomyces sp .), Streptomyces 8E12 cells, culture medium, extracts, methoxyhygromycin or epi-methoxyhygromycin isolated from it confirms the selective herbicidal activity and information about these biological activities Focusing on, it was confirmed that it has a useful effect as an eco-friendly biopesticide.

Figure 1 is a photograph showing the spherical cylindrical spores of the genus 8E12 (KCTC 10336BP) of the new strain Streptomyces.

Figure 2 is a molecular weight analysis by SI-MS of methoxy hygromycin isolated from the culture strain of 8E12 (KCTC 10336BP) genus Strain strain Streptomyces.

Figure 3 is a figure showing a hydrogen-nuclear magnetic resonance ( ¹ H-NMR) photograph of methoxyhygromycin.

Figure 4 is a figure showing the carbon-nuclear magnetic resonance ( ¹³ C-NMR) photograph of methoxyhygromycin.

Fig. 5 shows selective herbicidal activity and bleaching symptoms of methoxy hygromycin against the leek [treatment concentration from left to right per pot (2 repetitions), 16 mg, 8 mg, 4 mg, 1 mg, 0.25 mg. , 0.1 mg, experimental results after 2 weeks of treatment].

Claims (6)

Streptomyces sp. 8E12 [KCTC 10336BP] strain, characterized by having herbicidal activity. A herbicide comprising Streptomyces spp. 8E12 [KCTC 10336BP] culture medium or extract and an agriculturally acceptable carrier. A herbicide comprising methoxyhygromycin or epi-methoxyhygromycin and an agriculturally acceptable carrier. delete A medium containing glucose, yeast extract, peptone, beef broth, and batamine is adsorbed on perlite and sterilized, followed by inoculation with 8E12 [KCTC 10336BP] strain of Streptomyces to incubate and culture. Culture method. 1) removing the cells from the culture solution of 8E12 [KCTC 10336BP] genus Streptomyces and obtaining a culture supernatant; 2) adsorbing the culture supernatant to an activated carbon column and then eluting the active fraction with acetone to concentrate under reduced pressure; 3) extracting the concentrate with butanol, concentrating the butanol layer under reduced pressure, and developing a mixture of chloroform and methanol on a silica gel column to obtain an active fraction; And 4) A method for separating methoxyhygromycin and epi-methoxyhygromycin, the step of concentrating the active fraction under reduced pressure, and separating it on column chromatography with methanol and analyzing on HPLC.