KR102110875B1 - Composition for controlling pine wilt disease comprising Streptomyces sp. AE170027 or compound isolated therefrom as effective component - Google Patents

Abstract

The present invention relates to a composition for controlling pine wilt disease comprising Streptomyces sp. AE170027 strain or a teleocidin B4 ((5S,6Z,8E,10E,12R,14Z)-5,12-dihydroxyicosa-6,8,10,14-tetraenoic acid) compound isolated therefrom as an effective component. Streptomyces sp. AE170027 strain or teleocidin B4 isolated therefrom may provide an environment-friendly biotic pesticide effective in controlling pine wilt disease and thus is expected to be very useful in related industry.

Description

A composition for controlling pine wilt disease comprising Streptomyces sp. AE170027 strain or a compound separated from the strain as an active ingredient {Composition for controlling pine wilt disease comprising Streptomyces sp. AE170027 or compound isolated therefrom as effective component}

The present invention relates to a composition for controlling pine wilt disease, comprising a strain of Streptomyces sp. AE170027 or a compound isolated from the strain as an active ingredient.

Pine wood disease (Pine wood disease), caused by Bursaphelenchus xylophilus , began in Japanese pine forests from the beginning of the 20th century and is currently raising concerns worldwide. In the pine tree nematode, 38 species of Pinus sp. Are known as hosts worldwide, and they cannot move on their own, but they migrate through the insect worm Monochamus alternatus to infect pine trees. When the pine tree nematode in the infected tree escapes by the fable of the pine tree celestial larvae, it invades the body of the insect, escapes together, and rapidly grows into the pine tree. In Korea, large-scale damage is currently occurring in the Gyeongnam region, and other damages are also spreading to Jeju, Jeonnam, Gangneung, and Gyeonggi-do regions.

Currently, as a method for controlling pine wilt disease, a method of controlling pine wilt disease by spraying a tree with a compound having an insecticidal action or a compound having insecticidal activity is used. However, there is a disadvantage that the work is difficult, and the drugs for water pipe injection are expensive and expensive. Therefore, research has been conducted on compounds having an effective nematode effect.

In the present invention, the nematode activity against pine wilt was identified from Actinomyces isolated from soil, and the chemical structure was determined by separating and purifying the active material produced by Actinomycetes.

On the other hand, Korean Patent No. 1501033 discloses' Streptomyces Goyangensis BIG11003 strain having a nematode effect and its use ', and Korean Patent No. 1855264 describes' Pine wiltworm containing pimprinetin as an active ingredient. Bottle control composition 'is disclosed, but there is no description of a composition for controlling pine wilt disease, which contains a strain of Streptomyces sp. AE170027 of the present invention or a compound isolated from the strain as an active ingredient.

The present invention was derived by the above-mentioned needs, and the present inventors isolated a strain of Actinomycetes AE170027 showing excellent nematode activity against pine wilt disease from a pine tree in Gangwon-do, and as a result of 16S rRNA analysis, the actinomycetes are Streptomyces blast myceticus ( Streptomyces blastmyceticus ) and 99.93% similarity. In addition, in order to confirm the active ingredient showing nematode activity in the strain, as a result of performing ¹ H-NMR, ¹³ C-NMR, HMQC, ¹ H- ¹ H COSY, and HMBC spectrum analysis, AE170027 strain By confirming that the active substance is teleocidin B4 (teleocidin B4; (5S, 6Z, 8E, 10E, 12R, 14Z) -5,12-dihydroxyicosa-6,8,10,14-tetraenoic acid), the present invention Completed.

In order to solve the above problem, the present invention provides a strain of Streptomyces sp. AE170027 strain having a deposit number KCTC13793BP having a nematode activity against pine wilt.

In addition, the present invention is a teleocidin B4 represented by Formula 1 (teleocidin B4; (5S, 6Z, 8E, 10E, 12R, 14Z) -5,12-dihydroxyicosa-6,8,10,14-tetraenoic acid) compound , Streptomyces sp. AE170027 strain strain culture medium having an agrochemically acceptable salt or a teleosidin B4 compound represented by Formula 1 and having a deposit number KCTC13793BP, as an active ingredient, pine wilt nematode Provided is a composition for controlling a bottle.

In the present invention, Streptomyces sp., A compound isolated from the AE170027 strain, teleocidin B4 (teleocidin B4; (5S, 6Z, 8E, 10E, 12R, 14Z) -5,12-dihydroxyicosa-6,8 , 10,14-tetraenoic acid) was confirmed that the nematode activity against pine wilt ( Bursaphelenchus xylophilus ) is about 1.3 times higher than that of abamectin known as a pine wilt control agent. Therefore, the strain AE170027 of the genus Streptomyces of the present invention and teleosidin B4 isolated therefrom are expected to be very useful in related industries because they can provide an environmentally friendly biopesticide effective in controlling pine wilt disease.

Figure 1 shows the step of confirming the effect of the nematode nematode on a 96-well plate targeting the strains isolated in the present invention.
2 is a schematic diagram showing the separation and purification process of AC-18-24.
3 shows the HPLC profile of AC-18-24.
4 shows the ESI-Mass spectrum of AC-18-24.
5 shows the ¹ H-NMR spectrum of AC-18-24.
6 shows the ¹³ C-NMR spectrum of AC-18-24.
7 shows the HMQC spectrum of AC-18-24.
Fig. 8 shows the ¹ H- ¹ H COSY spectrum of AC-18-24.
9 shows the HMBC spectrum of AC-18-24.
Figure 10 shows the HMBC and ¹ H- ¹ H COSY correlation of AC-18-24.
11 shows the chemical structure of AC-18-24.

In order to achieve the object of the present invention, the present invention provides a strain AE170027 of Streptomyces sp. Having a deposit number KCTC13793BP having a nematode activity against pine wilt.

The strain AE170027 of the genus Streptomyces was isolated from pine trees, and has a nematode activity against pine wilt ( Bursaphelenchus xylophilus ), telecydin B4 (teleocidin B4; (5S, 6Z, 8E, 10E, 12R, 14Z) -5, 12-dihydroxyicosa-6,8,10,14-tetraenoic acid ) in a strain which can produce, the genus Streptomyces (Streptomyces sp.) AE170027 strain was deposited with 117 of January 2019 in biological resource Center (Accession Number: KCTC13793BP).

In addition, the present invention, a teleocidin B4 represented by the following Chemical Formula 1 (teleocidin B4; (5S, 6Z, 8E, 10E, 12R, 14Z) -5,12-dihydroxyicosa-6,8,10,14-tetraenoic acid) A pine tree of a plant containing a culture medium of Streptomyces sp. AE170027 strain having an accession number KCTC13793BP containing a compound, agrochemically acceptable salt thereof, or a teleosidin B4 compound represented by the following Chemical Formula 1 as an active ingredient Provided is a composition for controlling nematode disease.

In the composition for controlling pine nematodes according to the present invention, the 'culturing solution' of the AE170027 strain of the genus Streptomyces having the accession number KCTC13793BP may include a culture medium of the strain, a supernatant of the culture medium, or a dried product thereof.

The composition for controlling pine wilt disease of the present invention may be prepared in the form of a directly sprayable solution, powder and suspension, or in a highly concentrated aqueous, oily or other suspension, dispersion, emulsion, oily dispersion, paste, dust, dispersion material or granule, , But is not limited to this.

The composition for controlling pine wilt disease of the present invention is a powder, pellet or granule to contain a culture medium of a cultured cell, freeze-dried cell and starch, crude protein and rock powder, etc. , Can be used as it is formulated with microcapsules. The composition is obtained by adding other ingredients that are easy to apply depending on the nematodes and crops, such as surface activators, inert carriers, preservatives, wetting agents, accelerators, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectors, buffers, etc. can do. The composition of the present invention may be in a form or concentrate or primary composition suitable for direct application by dilution with an appropriate amount of water or other diluent prior to application. It will be clearly understood by those skilled in the art that nematode concentrations may vary, particularly when concentrated or used directly, depending on the nature of the particular agent, the type of plant applied, the soil and climate of the cultivation area, and the like.

In addition, the composition for controlling pine wilt disease of the present invention may further include an agriculturally acceptable carrier. Suitable solid carrier materials are, in principle, all porous, agriculturally acceptable carriers such as mineral earths (e.g. silica, silica gel, silicate, talc, kaolin, limestone, lime, choke, bowl, ocher, clay, Dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, pulverized synthetic materials, fertilizers (eg ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), vegetable products (eg grain flour, bark flour, wood meal) And nut shell powder) or cellulose powder, but is not limited thereto. Further, the solid carrier may be used by mixing one kind or two or more kinds.

In addition, the composition for controlling pine wilt disease of the present invention may further contain a nematode active material, and the nematode active material is not limited thereto, but avermectin-based nematode active material, morantel tartrate (morantel tartrate), may be one or more selected from the group consisting of mesulfenfos (mesulfenfos) and levamisol (levamisol). The avermectin-based nematode active material may be emamectin benzoate, abamectin, milbemectin or ivermectin, but is not limited thereto.

In the composition according to an embodiment of the present invention, the plant may be a plant of the genus Pine, fir, spruce or oak, but is not limited thereto.

The present invention also provides a method for preventing or controlling pine wilt disease of a plant comprising the step of treating the composition for controlling pine wilt disease in plants, seeds or plantations of plants.

In the method for preventing or treating pine wilt disease of the present invention, the composition for controlling pine wilt disease is as described above.

In the method for preventing or treating pine wilt disease of the present invention, the composition for controlling pine wilt disease starts or appears as a preventive protective measure before pine wilt disease appears, or cultivation of these plants The damage caused by pine nematodes can be suppressed or reduced by transferring or spraying it on soil or seeds.

The method of the present invention is not limited as long as it is a tree in which pine wilt disease control is required, and for example, the composition of the present invention can be treated on a plantation of pine, fir, spruce or oak. As the treatment method, a method known in the art, such as immersion treatment, soil irrigation treatment, or foliar spray treatment, may be used.

The present invention also provides a method for preparing a composition for controlling pine wilt disease of plants, comprising culturing a strain of Streptomyces sp. AE170027 having a deposit number of KCTC13793BP.

The strain AE170027 of Streptomyces genus having a deposit number of KCTC13793BP of the present invention shows teleecidin B4 (teleocidin B4; (5S, 6Z, 8E, 10E, 12R, 14Z)-which shows excellent nematode activity against pine wilt ( Bursaphelenchus xylophilus )) 5,12-dihydroxyicosa-6,8,10,14-tetraenoic acid) compound can be produced, and thus, when the strain of the present invention is cultured, a composition for controlling pine wilt disease can be prepared.

The method for culturing the strain of the present invention can be cultivated according to a method commonly used in the art, and a method for separating and purifying the teleosidin B4 compound from the culture medium of the strain is also performed according to a method commonly used in the art. Can be performed.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

1. Actinomycetes separation, culture and identification

Actinomycetes used in the invention are HV agar (Humic acid-Vitamin agar) medium (0.1% humic acid: dissolved in 0.2N NaOH, 0.05% Na ₂ HPO ₄ , 0.171% KCl, 0.005% MgSO ₄ , 0.001% from pine trees in Pyeongchang, Gangwon-do) FeSO ₄ · 7H ₂ O, 0.002% CaCO ₃ , 50 ppm cycloheximide, 10 ppm nalidixic acid, 8 kinds of vitamins including thimine-HCl and 2% agar) were isolated. Identification of the isolated strain was analyzed using 16S rRNA base sequence. To preserve isolated Actinomycetes, it was cultured in Bennett's medium (1% glucose, 0.1% yeast extract, 0.2% Bacto-peptone, 0.1% beef extract). In addition, the culture for the evaluation of nematode activity of Actinomycetes is GSS medium (1% soluble starch, 2% glucose, 2.5% soybean meal, 0.1% beef extract, 0.4% yeast extract, 0.2% NaCl, 0.025% K ₂ at 28 ° C). HPO ₄ and 0.2% CaCO ₃ ) were used. To screen for nematode activity, an equal amount of acetone was added to 0.5 ml of the culture medium, shaken at room temperature, and ultrasonically extracted for 15 minutes. After centrifugation, 0.2 ml of supernatant was concentrated, dissolved in 50 μl of DMSO, and used for nematode activity experiment.

2. Selection of active strains of nematodes

The nematode activity test for isolated strains is as follows. The pine nematode ( Bursaphelenchus xylophilus ), which is the cause of pine nematode disease , was pre-sold at the National Forest Research Institute and subcultured on a plate plate of PDA (Potato Dextrose Agar) cultured with fungus ( Botrytis cinerea ), which was used as a test nematode. For the fungal nematode effect, 5 μl of Actinomycetes culture extract and 95 μl of nematode solution (including 30 to 40 nematodes) were tested in duplicate in each well of a 96-well Microtest ™ Tissue Culture Plate. For nematode activity, a 96-well plate treated with a culture solution was placed in a 25 ° C incubator and reacted. After 24 hours, the stiff and immovable nematodes under a dissecting microscope were considered as dead nematodes, and flexible and mobile nematodes were considered as acid nematodes. The nematode rate was investigated.

Criteria for the determination of the activity of acaricidal nematodes Active criteria Death rate (%) One 0 1.5 20-40 2 40-75 2.5 75-95 3 100

Example 1. Selection and identification of active strains of nematodes

As a result of the nematode activity test on the isolates, strain AE170027, which exhibited more than 70% of nematodes at 1-2 mg / ml after 24 hours of treatment with extracts of 0.5-2 mg / ml, was selected. As a result of molecular phylogenetic analysis based on 16S rRNA gene sequence, AE170027 strain showed 99.93% similarity with Streptomyces blastmyceticus .

Screening results of active fungal nematodes strain Scientific name Mg / ml activity AE170027 Streptomyces sp. 2 3 One 2 0.5 1.5

Example  2. From the AE170027 strain Pine wilt  Separation and purification of control substances

2-1. Separation and purification of pine wilt control material

AE170027 The culture medium was centrifuged at 5,000 rpm for 30 minutes to separate into a broth layer and a mycelium cake layer. As a result of confirming that the extract obtained by fractionating the Mycelium cake layer into various solvents according to polar and non-polar solvent conditions has control activity through bioassay, it showed activity in a hexane layer (991 mg) and the sample was silica- Gel thin layer chromatography (TLC) was used to characterize the material. After that, in order to purify the hexane layer, silica-gel column chromatography was carried out in a step-wise gradient method using CHCl ₃ : MeOH = (50: 1 ~ 1: 1) developing solvent, and based on the results, activity was shown. The fraction of CHCl ₃ : MeOH = 50: 1 (895 mg) was separated by silica-gel column chromatography in a linear gradient using CHCl ₃ : MeOH = 50: 1 developing solvent to obtain a fraction (165 mg). ). Subsequently, a size-exclusion gel, Sephadex LH-20, was subjected to column chromatography to obtain a fraction having a control activity (39.2 mg). The fraction was finally subjected to a high performance liquid chromatography (HPLC) step to separate and purify a substance having a control activity (FIG. 2).

As a result of checking by HPLC, the peak of the active material was confirmed at about 17 minutes when the mobile phase was flowed with 85% methanol at 254 nm, and it was confirmed that the separated material was a single material (FIG. 3). The present inventors confirmed the control activity of this material, named it AC-18-24, and conducted a NMR (nuclear magnetic resonance) analysis to determine the chemical structure.

2-2. Molecular weight measurement of AC-18-24

To measure the molecular weight of AC-18-24, the ESI-Mass spectrum was measured in positive mode. In the positive mode, [M + H] ⁺ was observed at m / z 452.3, indicating that the molecular weight was 451, and the molecular formula was determined to be C ₂₈ H ₄₁ N ₃ O ₂ (FIG. 4).

2-3. ¹ H-NMR and ¹³ C-NMR spectrum of the AC-18-24

AC-18-24 ¹ H-NMR (Fig. 5) and ¹³ C-NMR (Fig. 6) AC-18-24 through a was found to be 28 carbons and 41 protons (proton) is present. As a result of measuring the ¹ H-NMR spectrum as shown in FIG. 5, amino protons were observed at 8.66 ppm, amino carbonyl protons were observed at 7.46 ppm, and 1 hydroxy at 1.24 ppm. ) Protons were observed. In addition, as a result of measuring ¹³ C-NMR spectrum as shown in FIG. 6, 6 aromatic carbons between 146.0 and 106.2 ppm and 4 olefin carbons at 151.9, 111.3, 120.8, and 114.0 ppm, at 174.5 ppm One aminocarmonyl proton was observed and found to be probable with ¹ H-NMR. The chemical shift of protons and carbons was confirmed through ¹ H-NMR and ¹³ C-NMR spectral analysis and summarized in Table 3.

2-4. 2D NMR spectrum of AC-18-24

As a result of measuring the heteronuclear multiple quantum coherence (HMQC) spectrum (FIG. 7) in order to confirm the link between the proton and carbon of AC-18-24, H-2 (d _H 6.77) and C-2 (d _C 120.8) , H-5 (d _H 6.49) and C-5 (d _C 106.2), H-8 (d _H 2.99, 3.10) and C-8 (d _C 33.8), H-9 (d _H 4.32) and C- 9 (d _C 55.9), H-12 (d _H 4.30) and C-12 (d _C 70.7), H-14 (d _H 3.71, 3.53) and C-14 (d _C 65.0), H-20 (d _H 1.50) and C-20 (d _C 21.5), H-29 (d _H 1.34) and C-29 (d _C 29.1), H-17 (d _H 0.90) and C-17 (d _C 21.6), H -15 (d _H 2.59) and C-15 (d _C 28.4), H-18 (d _H 2.89) and C-18 (d _C 32.9), H-21 (d _H 6.15) and C-21 (d _C 151.9), H-22 (d _H 5.40, 5.24) and C-22 (d _C 111.3), H-23 (d _H 1.89) and C-23 (d _C 34.8), H-24 (d _H 1.89) C-24 (d _C 24.9), H-26 (d _H 2.24) and C-26 (d _C 37.9), H-23 (d _H 1.43) and C-23 (d _C 34.8), H-24 (d _H 1.40) and C-24 (d _C 24.9), H-16 (d _H 0.68) and C-16 (d _C 19.6), H-28 (d _H 0.53) and C-28 (d _C 18.0) Was confirmed. In addition, ¹ H- ¹ H COSY spectrum (FIG. 8) ^first cross peak between H ¹ H- (cross peak) to confirm the results, links, of 7.46 8.66ppm (H-1) and 6.77ppm (H-2) through ppm (H-10) and 4.32 ppm (H-9) linkage, 4.32 ppm (H-9) and 2.99 ppm (H-8), 3.10 ppm (H-8), 3.71 ppm (H-14), 3.53ppm (H-14) and 6.77ppm (H-2) and 2.99ppm (H-8), 3.10ppm (H-8) and 4.30ppm (H-12) and 2.59ppm (H) -15) and 2.59ppm (H-15) and 0.68ppm (H-16), 0.90ppm (H-17) and 2.24ppm (H-26) and 0.53ppm (H-28), 1.00ppm (H-27) and 1.89ppm (H-23) and 1.40ppm (H-24) and 1.89ppm (H-24) and 1.43ppm (H-23) Could. Finally, as a result of measuring the heteronuclear multiple bond correlation (HMBC) spectrum (FIG. 9) to confirm the position of carbon in 2, 3, 4 bonds from protons, C-2 (from H-1 (d _H 8.66) d _C 120.8), H-3 (d _H 114.0), C-4a (d _C 116.8), H-7a (d _H 137.8), H-2 (d _H 6.77) to C-3 (d _C 114.0), C-4a (d _C 116.8), C-7a (d _C 137.8), C-8 (d _C 33.8), H-8 (d _H 2.99, 3.10) to C-2 (d _C 120.8), C-3 (d _C 114.0), C-4a (d _C 116.8), NH-10 (d _H 7.46) to C-8 (d _C 33.8), C-11 (d _C 174.5), C-12 (d _C 70.7) , C-14 (d _C 65.0), H-12 (d _H 4.30) to C-11 (d _C 174.5), C-4 (d _C 146.0), H-18 (d _H 2.89) to C-4 ( d _C 146.0), C-12 (d _C 70.7), H-5 (d _H 6.49) to C-4 (d _C 146.0), C-4a (d _C 116.8), C-7 (d _C 118.0), C-25 (d _C 40.0), H-26 (d _H 2.24) to C-24 (d _C 24.9), H-27 (d _H 1.00) to C-25 (d _C 40.0), H-28 (d _H 0.53) to C-25 (d _C 40.0), H-29 (d _H 1.34) to C-6 (d _C 138.6), C-24 (d _C 24.9), C-25 (d _C 40.0), C -26 (d _C 37.9), H-20 (d _H 1.50) to C-7 (d _C 118.0), C-19 (d _C 39.6), C-2 Long range coupling from 3 (d _C 34.8), H-21 (d _H 6.15) to C-19 (d _C 39.6), H-22 (d _H 5.40, 5.24) to C-19 (d _C 39.6) It was observed, and it was confirmed that it was a teleocitin B4 structure.

2-5. Chemical structure of AC-18-24

To determine the chemical structure of AC-18-24, ¹ H-NMR, ¹³ C-NMR, HMQC, ¹ H- ¹ H COSY, and HMBC spectra were analyzed through analysis of the structure, resulting in final separation. The chemical structure of AC-18-24 was identified as (5S, 6Z, 8E, 10E, 12R, 14Z) -5,12-dihydroxyicosa-6,8,10,14-tetraenoic acid (FIG. 11).

Example  3. Of the material purified from the AE170027 strain Nematode activity  evaluation

In order to confirm the control effect of AC-18-24 isolated and purified from the AE170027 strain against the pine wilt disease, an experiment was conducted to evaluate the nematode activity. Abamectin was used as a positive control for the evaluation of nematode nematode activity, and the activity was compared with AC-18-24, a purified substance. The test method was carried out in the same manner as the screening method for the active strain of the nematode, and as a result, the half-lethal dose (LC ₅₀ ) value of abamectin was confirmed to be 4.1 μg / ml, and the LC ₅₀ value of AC-18-24 was 3.125 μg / ml. It was confirmed that the nematode activity of AC-18-24 of the present invention was about 1.3 times higher than that of abamectin (Table 4).

Nematode activity of AC-18-24 (Teleoxidin B4) sample LC ₅₀ (µg / ml) Abamectin 4.1 AC-18-24 3.125

Korea Research Institute of Bioscience and Biotechnology KCTC13793BP 20190117

Claims (6)

Streptomyces sp. AE170027 strain with deposit number KCTC13793BP, which has nematode activity against pine wilt. Teleocidin B4 represented by Formula 1 below (teleocidin B4; (5S, 6Z, 8E, 10E, 12R, 14Z) -5,12-dihydroxyicosa-6,8,10,14-tetraenoic acid) compound, agrochemical thereof A composition for controlling pine wilt disease of a plant containing a culture medium of Streptomyces sp. AE170027 having an acceptable salt or a teleosidin B4 compound represented by the following Chemical Formula 1 and having a deposit number KCTC13793BP as an active ingredient .
[Formula 1]

3. The composition according to claim 2, wherein the plants are pine, fir, spruce or leaf. The composition according to claim 2, further comprising a nematode active material. The method of claim 4, wherein the nematode active material is selected from the group consisting of avermectin-based nematode active material, morantel tartrate, mesulfenfos, and levamisol. A composition characterized by one or more. The composition of claim 5, wherein the avermectin-based nematode active material is emamectin benzoate, abamectin, milbemectin or ivermectin. .

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