KR101666676B1 - Streptomyces netropsis AN110065 strain having nematocidal activity against root-knot nematode and uses thereof - Google Patents

Abstract

The present invention is Streptomyces net drop system (Streptomyces having a nematocide activity against root-knot nematode The present invention relates to a microorganism preparation for the nematode root nematode, which comprises the above-mentioned strain, spore, cultivar or cultured extract as an active ingredient, and a microorganism preparation for the nematode of the root-knot nematode in a crop, And a step of culturing the strain. The present invention also provides a method for preparing a microorganism preparation for a nematode of a root-knot nematode comprising the step of cultivating the microorganism.

Description

Streptomyces nunlopsis strain AN110065 having domesticated nematode activity against root-knot nematodes and its use {Streptomyces netropsis AN110065 strain having nematode activity against root-knot nematode and uses thereof}

The present invention is Streptomyces net drop cis AN110065 strain and relates to the use thereof, more specifically, Streptomyces net drop system (Streptomyces having a nematocide activity against root-knot nematode with the nematocide activity against root-knot nematode The present invention relates to a microorganism preparation for the nematode root nematode, which comprises the above-mentioned strain, spore, cultivar or cultured extract as an active ingredient, and a microorganism preparation for the nematode of the root-knot nematode in a crop, And a step of culturing the strain. The present invention also provides a method for preparing a microorganism preparation for a nematode of a root-knot nematode comprising the step of cultivating the microorganism.

Meloidogyne spp . , A member of the plant parasitic nematode, is absolutely parasitic on plant roots and causes serious damage to important crops. Globally, about 11% of crop damage is known to be caused by nematodes, and when converted to money, crop losses amount to $ 1.18 trillion a year. In Korea, it was reported that about 54% of the paddy fields of Saju County in Gyeongbuk Province were infected with root-knot nematode. Four important nematodes in agriculture are Meloidogyne incognita ), carrot root nematode ( Meloidogyne hapla ), Java root nematode ( Meloidogyne javanica ), and peanut nematode ( Meloidogyne arenaria ). Among them, a root-knot nematode, which is a problem in domestic plantation, is meloidogyne incognita and Meloidogyne < RTI ID = 0.0 > arenaria . Among the economically important vegetable crops, tomatoes ( Solanum lycopersicum L.) has a large planting area and production rate, and it is mainly caused by the cultivation of the plant. Therefore, the occurrence of root-knot nematode is a major problem due to the cultivation of the crop, and it is known that the nematode to which tomato is added is meloidogine incognita. have.

Synthetic pesticides were generally used to control root-knot nematodes. In recent years, however, there has been increasing interest in safe food around the world and the use of toxic nematocides has become increasingly strict due to policies to maintain agriculture in a more environmentally safe manner. In Europe, the preparation and use of methyl bromide, which is mainly used as soil fumigant due to ozone layer destruction and environmental pollution problems, will be prohibited from 2015. Also, cadusafos as a non-fumigant pesticide, Will be prohibited. Therefore, it is urgently required to develop a safer nematicide, which has a safer and more effective effect on the environment that can replace it, and many researchers are carrying out a lot of research to develop a biosarin nematide using plants and microorganisms.

In recent years, a method of using microorganisms as an environmentally friendly method for controlling root-knot nematodes has been studied. It has been studied that various microorganisms produce various secondary metabolites and have various antibacterial and insecticidal activities by directly introducing pathogens (Zhao et al. (2014) Antagonistic action of Bacillus subtillis strain SG6 on Fusarium graminearum. PLoS one. 9 (3), E92486). Therefore, studies are underway to select strains that have activity through screening of live nematode for various microbial cultures, and to develop strains that control root blight nematodes using them.

Korean Patent No. 1101783 discloses a novel strain of Streptomyces sp. Having excellent antimicrobial activity and insecticidal activity, a method of using the same, and a culture using the culture method. In Korean Patent Publication No. 2010-0116562, A strain of Streptomyces crispus G341 and a method of controlling plant disease using the strain of Streptomyces crispus G341 have been disclosed. However, as in the present invention, Streptomyces nernopicus strains AN110065 having nematicidal activity against root-knot nematodes and their uses have never been disclosed.

The present invention has been made in view of the above-mentioned needs. The present inventor has continued research on the tomato root rot nematode in order to find not only the control effect in packaging but also the biological control agent having utility value as a commercial pesticide , And Streptomyces nunlopsis strain AN110065 isolated from grassland soil of Pyeongchang - gun had the best nematicide activity among 2,700 actinomycetes. Therefore, the inventive strain can be used as a biological pesticide for the control of root-knot nematode, and can be provided as an innovative biological control agent capable of replacing chemical pesticides and preventing problems caused by environmental pollution. Completed.

In order to solve the above problems, the present invention provides Streptomyces netropsis strain AN110065 having nematode activity against root-knot nematodes.

In addition, the present invention provides a microorganism preparation for nematode against a root-knot nematode comprising the strain, spore, culture or cultured extract as an active ingredient.

In addition, the present invention provides a method for controlling root-knot nematodes comprising treating a root-knot nematode microorganism preparation to a crop, a seed of a crop, or a cultivated land.

In addition, the present invention provides a method for producing a microorganism preparation for a nematode against a root-knot nematode comprising the step of culturing the strain.

Streptomyces net drop system (Streptomyces according to the invention netropsis) AN110065 strains in vitro (in vitro ), as well as in tomato root nodule nematode, it is useful for the control of crop root nematode disease by showing concentration - dependent nematode activity.

In particular, in this strain, the culture filtrate has no effect, but only the cell extract is effective. Since the nematicide active substance is not secreted into the culture filtrate and is present in the cell, the active substance is recovered in order to commercialize the microbial nematode There is a big advantage. That is, it shows that the microbial nematode exhibiting high activity can be developed by collecting only the cells. Thus Streptomyces net drop system (Streptomyces netropsis ) AN110065 is highly likely to develop a new microbial nematode product.

1 is a Streptomyces net drop system (Streptomyces netropsis ) AN110065 strain of root-knot nematode.
Figure 2 is a graph showing the activity of Streptomyces < RTI ID = 0.0 > netropsis) it is a scanning electron microphotograph (B) 14-day state of the cultured embryoid bodies (A) and the spore surface AN110065 strain 28 ℃.
Figure 3 is a graph showing the activity of Streptomyces < RTI ID = 0.0 > The phylogenetic tree is based on the nucleotide sequence of 16S rRNA of the netropsis AN110065 strain.
Figure 4 is a graph showing the activity of Streptomyces < RTI ID = 0.0 > netropsis ) AN110065 was obtained from the solvent fraction obtained from the three fractions obtained in the solvent fraction.
5 is Streptomyces net drop system on tomato root-knot nematode disease (Streptomyces netropsis AN110065 culture medium (A) and the control effect (B) of the treatment zone.

In order to achieve the above object, the present invention provides Streptomyces netropsis strain AN110065 having nematode activity against root-knot nematodes.

Streptomyces net drop system (Streptomyces according to the invention netropsis ) AN110065 is a naturally occurring microorganism in Korea and is characterized by having nematode activity against root-knot nematode of various crops.

More specifically, Streptomyces netropsis strain AN110065 of the present invention was isolated from grassland soil and formed white spores on Bennett agar medium. As a result of observation with a scanning electron microscope, a rectiflexible sporangia And form spores with a cylindrical shape of 0.4 to 1.2 mu m in size.

In addition, 16S rRNA molecular phylogenetic analysis based on the gene sequence, AN110065 strain Streptomyces net drop system (Streptomyces netropsis ) And showed 99.78% similarity (EZ-taxon blasting) to NBRC 3723. The strain thus isolated was designated as Streptomyces netropsis AN110065 strain and deposited with the International Depository Agency (Korea Research Institute of Bioscience and Biotechnology) on September 23, 2013 and assigned the deposit number KCTC 12490BP .

In a strain according to an embodiment of the present invention, the root-knot nematode is selected from the group consisting of Meloidogyne incognita ), carrot root nematode ( Meloidogyne hapla ), Java rootstock nematode ( Meloidogyne javanica ), peanut nematode ( Meloidogyne arenaria ) and tomato root nodule ( Meloidogyne incognita , and the like.

In a strain according to one embodiment of the present invention, the crop may be, but is not limited to, tomato.

The present invention also relates to a method for screening for a nematode of a root-knot nematode containing Streptomyces netropsis strain AN110065 (accession number: KCTC 12490BP), a bacterium of the strain, a spore, a cultured product or a culture extract as an active ingredient A microbial agent for a microorganism.

The microorganism preparation may include a medium in addition to Streptomyces netropsis AN110065 strain (accession number: KCTC 12490BP), a microorganism, a spore, a culture, or an extract of the microorganism, and the medium may include bran, Etc. can be used.

In addition, the microorganism preparation may be a powder, a pellet, a granule or a granule using a culture obtained by culturing the strain of Streptomyces netropsis strain AN110065 in a bran or bran / Solution or the like.

In the present invention, the microbial agent further comprises lime. More specifically the Streptomyces net drop system (Streptomyces netropsis) AN110065 strain (Accession No: KCTC 12490BP), the strain cells, spores, cultures, and lime with effectiveness minutes and at least one selected from the culture (CaO 60%, MgO 10% , handling a mixture of SiO 2%) As a result, the control effect was high irrespective of the treatment time of the active ingredient.

In addition, at least one effective ingredient selected from strains of Streptomyces netropsis AN110065 (accession number: KCTC 12490BP), cells of the strain, spores, cultures and cultures is treated at the time of seeding, Was found to be highly effective in the treatment of molds at the time of molding.

In the microorganism preparation of the present invention, the Streptomyces net drop system (Streptomyces netropsis) AN110065 strain or one to variously modified by per se known methods used in the art for the culture thereof in the form of liquid and powdered can be used, preferably Streptomyces net drop system (Streptomyces netropsis ) AN110065 is adsorbed and dried on a carrier such as starch, crude protein or rock fractions. The carrier which can be used by mixing with the culture broth or concentrate of the strain of the present invention may be any carrier used in the art. Specific examples of the carrier that can be used in the present invention include seeds such as rice, wheat, corn, barley, soybeans, sorghum, millet, millet and the like, cereals such as cereals and potatoes, rootstocks such as sweet potatoes and cassava, (For example, powder), starches derived therefrom and derivatives thereof, and the like. In addition, diatomaceous substances such as agar, gelatin, pectate (polygalacturononate), chitosan, carboxymethylcellulose and derivatives thereof, clay minerals such as gelite, natural wax, natural gum, kaolin and bentonite, . These various carriers may be used individually, or a carrier of improved physical properties may be obtained by mixing two or more carriers in a suitable ratio. When such carriers are used, they can be metabolized by microorganisms and become a nutrient source. These carriers are highly viscous and thus increase the adhesiveness to the surface of plants.

The present invention can also include a dry product obtained by a drying method and a biological pesticide containing the strain or strain culture solution. The dried product can be prepared into a formulation selected from the group consisting of a wetting agent (WP), a solid phase (GM), a granular wettable agent (WG), a granule (GR), a powdered (DP) have. These bio-pesticide formulations are superior to conventional liquid formulations in terms of stability and physico-chemical properties and can be used for plant disease control.

A wetting agent in a biocidal pesticide refers to a formulation in which the pesticide is hydrated when diluted in water as a powder and the solid phase is a formulation in which a microorganism culture fluid is mixed with or adsorbed on a solid phase material and is not classified as a powder, granule or wettable powder. The granular wettable powders may be formulated in the form of a pesticide used as a granular phase by diluting with water, a granular form of the pesticide used as a granule used as a granule, a granule of a pesticide used as a granule, Means a formulation of pesticide used as a suspension by hydration before treatment with seed as a powder.

Wettable powder according to one embodiment of the present invention are the active ingredient (active ingredient) as Streptomyces net drop system (Streptomyces netropsis AN110065 strain, white carbon as a desiccant, sodium bis [2-ethylhexyl] sulfosuccinate as a wetting agent, sodium lignosulfonate as a dispersing agent and kaolin as an extender, preferably 10 weight % Streptomyces net drop system (Streptomyces netopsis AN110065 strain, 1 wt% white carbon, 1 wt% sodium bis [2-ethylhexyl] sulfosuccinate, 1 wt% sodium lignosulfonate and 87 wt% kaolin.

The liquid wettable powder according to one embodiment of the present invention is used for controlling root-knot nematodes. In order to control the root-knot nematode by using the microorganism preparation of the present invention, the microorganism preparation can be uniformly diluted with water and then sprayed onto crops, seeds of crops or cultivated land using an appropriate spraying device such as a power sprayer.

In addition, the present invention provides a method for controlling root-knot nematodes comprising treating a root-knot nematode microorganism preparation to a crop, a seed of a crop, or a cultivated land.

The method of controlling the microbial agent may be, but not limited to, the treatment of the microbial agent upon sowing or planting of the crop.

More particularly, the present invention relates to a method for treating microbial agents, which comprises treating the microbial agent with i) the soil, ii) treating the soil with the soil, iii) mixing with the lime, iv) treating the solid cultured in bran / Or v) a combination of the above treatment methods can inhibit root-knot nematode of the crop.

In the present invention, the method of the present invention is a method of treating an effective component in a microorganism preparation by treating 0.1% to 10% (v / v) or treating a spore concentration of 1.0 x 10 ⁴ cells / ml to 5.0 x 10 ⁷ cells / . More preferably, the culture medium cultured from bran medium, wheat bran / vaginal medium and vaginal medium is used at a concentration of 1% to 5% (v / v) or at a concentration of 1.0 × 10 ⁵ cells / ml to 5.0 × 10 ⁶ cells / It can be applied to the soil before sowing of the crop or to the port before the planting, or to the pavement at the time of planting, but the present invention is not limited thereto.

The microbial agent may be used for an antimicrobial microbial pesticide, a seed coating agent, a microbial nutrient, a soil improving agent, a composting homework, a foliar spraying agent, or a foliar spraying agent or the like, including a culture solution prepared by culturing the above-mentioned strains under the above- have.

In addition, the present invention provides a method for producing a microorganism preparation for a nematode against a root-knot nematode comprising the step of culturing the strain. The Streptomyces net drop system (Streptomyces The method for culturing the strain of AN110065 and the method for producing the microorganism preparation may be any method known in the art and is not particularly limited to a specific method. Further, Streptomyces net drop system (Streptomyces of the invention In the case of netopsis AN110065 strain, the culture filtrate had no effect, but only the cell extract showed the effect, and it was confirmed that the nematicide active substance was present in the cells without being secreted by the culture filtrate. Therefore, when the culture obtained after culturing the strain of the present invention was prepared with an organic solvent, the effect of controlling the root-knot nematode was excellent when it was extracted with ethyl acetate.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Isolation of actinomycetes from soil

Actinomycetes were isolated from grassland soil of Pyeongchang - gun, Gangwon - do. 10 g of the soil sample collected in the sterilized water was suspended and stirred in an incubator at 28 ° C and 150 rpm for 30 minutes. Taking the stirred suspension 0.1 ㎖ humic acid (Humic acid) used as Streptomyces separation medium-vitamin agar (0.1% humic _{acid, 0.05% Na 2 HPO 4,} 0.171% KCl, 0.005% MgSO 4, 0.001% FeSO 4 · H _{2 O, 0.002% CaCO 3,} 2% agar) (Hayakawa and Nonomura, 1987, J. Ferment. after the cells were cultured at 28 ℃ incubator for 7 days plated on Tech, 65, 501-509). The isolated actinomycetes were grown in Bennett's agar (1% glucose, 0.1% yeast extract, 0.2% bovine-peptone, 0.1% beef extract, 2% agar) (Hesseltine et al., 1954 Ann. (1% soluble starch, 0.025% K ₂ HPO ₄ , 2.5% soybean meal, 0.1% beef extract, 0.4% sodium chloride solution) were added to 100 ml of sterilized GSS medium 500 ml baffled (0.2% NaCl) containing 0.2% NaCl, 2% glucose, 0.2% CaCO ₃ (pH 7.2) (Kim et al., 1989 Korean J. Biotechnol. Bioeng, 4, 162-166) baffled Erlenmeyer flasks at 150 rpm and 28 ° C for 7 days.

Example  2. Root-knot nematode Second generation  Nematode activity against larvae

A total of 2700 strains of actinomycetes were isolated in the same manner as in Example 1, and the nematode activity of the second instar larvae of tomato root nodule was examined. In order to select microorganisms with excellent nematicidal activity, 2,700 actinomycetes culture samples were obtained from the Korea Research Institute of Bioscience and Biotechnology.

The root-knot nematodes used in the experiment were isolated and identified by Hwang et al. (2014 Kor, J. Hort. Sci. Technol, 32, 217-226) 25 ± 5 ° C) and used for experiments. Soil, which was mixed with the same quantity of sterilized sand, was placed in a plastic pot of 9.5 cm in diameter and seeded with tomato seedlings cultivated for 3 weeks by sowing. After growing for another week, 10,000 eggs per day were inoculated to the eggs of root nematode and cultivated for 45 to 60 days.

Tomato roots were taken from heavily infested tomato root nodules, and the root nodule larvae were isolated from the nodules and oocytes in the roots. The separation of root-knot nematode larvae was carried out by Barker et al. (1985 Nematode population dynamics with emphasis on determining damage potential to crops, p. 135-148. In: KR Barker, CC, Carter, and JN Sasser on Meloidogyne. North Carolina State University, Raleigh, North Carolina, USA, 2, 135-148) were done by improving the way, wash the tomato roots infected with root-knot nematodes water cut to 1 cm in size into the shredders 0.5% sodium hypochlorite The sodium hypochlorite solution was soaked in the tomato roots and then pulverized for 60 seconds. The pulverized sample was sieved using a 65 mu m sieve, and eggs were collected in a 25 mu m sieve. The nematode eggs collected in 25 μm sieve were washed three times with sterilized water to remove the remaining sodium hypochlorite solution and the Baermann funnel method (Southey, 1986 Laboratory methods for work with plant and soil nematodes, Ministry of Agriculture Fisheries and Food. Majesty's Stationery Office, London, UK, 202).

The nematode activity screening of 2,700 actinomycetes culture extracts was performed on 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). 1 ml of the actinomycetes cultured as described above was taken, and the same volume of methanol was added, followed by sonication for 20 minutes. The culture supernatant was centrifuged at 13,000 rpm for 10 min at 4 ° C to obtain a supernatant. A nematode (J2 larva) suspension (about 70/100 μl) was placed in each well of a 96-well tissue culture plate and the strain extract was treated with 5, 10, 20% to give a final volume of 50 μl per well. As a control, abamectin was used at the level of 0.5 and 1 ㎍ / ㎖, and the untreated control was treated with sterilized water: methanol (1: 1, v / v) at a level of 20%. After the sample was treated, the 96-well microplate was stirred for 30 seconds to uniformly mix the samples. To prevent oxygen deficiency and drying of the nematode, the plate was placed in a plastic bottle having a relative humidity of 100% and stored in a 25-27 ° C incubator. The experiment was carried out three times and 24 hours and 72 hours after the sample treatment, the nematode that died straight from the moving nematode under an optical inversion microscope (Olympus CKX41, Tokyo, Japan) (Abbott, 1925, 1925, J. Eco. Entonol, 18, 265-267) was used to determine the mortality rate using Abbott's formula.

(%) = [Mortality rate of treatment - mortality rate of control] / (100 - mortality rate of treatment)] × 100.

Twenty - eight candidate actinomycetes were selected for primary mortality of 50% or more. AN110065 was selected as the best strain in the second experiment. The strain AN110065 showed 68.3% activity at 24 hours after treatment with 10% of the culture broth, 78.9% at 20% treatment and little activity at 5% treatment (FIG. 1). In addition, 73.2% of the cells were treated with 10% treatment after 72 hours of treatment and 94.2% of the cells were treated with 20% treatment (FIG. 1). On the other hand, AN110065 showed no activity when treated with only culture filtrate.

In the case of Streptomyces netropicus strain AN110065, there was no nematode activity in the culture filtrate, and the high concentration of live nematode activity was found in the culture extract. Therefore, it was determined that the AN120065 nematode activity was due to intracellular substances. In the case of intracellular substances, there is an advantage that there is almost no loss of material when cells are harvested and formulated because microbial pesticides contain substances in the cells as compared with extracellular substances. As a result, it becomes possible to develop microbial nematodes having high activity.

Example  3. Identification of isolated strains

The AN110065 strain isolated in Example 1 and selected through Example 2 was analyzed for its genetic characteristics through morphological and 16S rRNA base sequences.

(1) Morphological characteristics

AN110065 was cultured for 14 days in a 28 < 0 > C incubator using Bennett's agar medium. The spore morphology and size of the isolates were examined by scanning electron microscopy (SEM, JSM-6700F, JEOL Ltd., Japan). The AN110065 strain sample for observation was fixed with glutaraldehyde (2.5%, 0.1 M phosphate buffer, pH 7.0) and dehydrated with ethanol. After dehydrated cells were immersed in isoamyl acetate, isoamyl acetate was evaporated with liquid CO _2, and then gold foil was observed and observed with an electron microscope. The AN110065 strain formed white spores and it was confirmed by scanning electron microscopy that the spores were formed as rectiflexible spores and formed a cylindrical spore having a size of 0.4-1.2 탆 (FIGS. 2A and 2B).

(2) Genetic characteristics

Molecular phylogenetic analysis of selected strains was carried out by 16S rRNA sequencing. The AN110065 strain was shake-cultured in 5 ml Bennett's liquid medium at 28 DEG C and 150 rpm for 3 days. The culture was centrifuged at 4 ° C and 13,000 rpm for 10 minutes to obtain cells. DNA was obtained from the precipitated cells using Qiagen's DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal primer set (27F; 5'-AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 2), 1492R; 5'-GGTTACCTTGTTACGACTT-3' PCR) was performed. The nucleotide sequence (SEQ ID NO: 1) was compared with the reported strains using the EzTaxon-e server (http://eztaxone.ezbiocloud.net) and 16S rRNA gene sequence information of NCBI. For each nucleotide sequence, a MEGA 4.0 program based on the neuronal binding method was used to generate the genealogy (Saitou and Nei, 1987 Mol. Biol. Evol, 4, 406-425). 16S rRNA gene molecular phylogenetic analysis based on the nucleotide sequence, AN110065 strain Streptomyces net drop system (Streptomyces netropsis ) And showed 99.78% similarity (EZ-taxon blasting) to NBRC 3723 (FIG. 3). The strain thus isolated was designated Streptomyces netropsis strain AN110065 and deposited with the International Depositary on Sep. 23, 2013 and assigned the deposit number KCTC 12490BP.

Example  4. Streptomyces Netopsis ( Streptomyces netropsis ) AN110065  The solvent of the strain culture Of the fraction layer Nematode  activation

In order to test the activity of the nematode according to the solvent fraction of the culture solution, Streptomyces netropsis) a AN110065 strain GSS (1% soluble _{starch, 0.025% K 2 HPO 4,} 2.5% soybean meal, 0.1% beef extract, 0.4% yeast extract, 0.2% NaCl 2g / L, 2% glucose, 0.2% CaCO ₃ (pH 7.2)) (Kim et al., 1989 Korean J. Biotechnol. Bioeng, 4, 162-166) at 28 ° C and 150 rpm for 10 days. After culturing, the same amount of methanol was added to 500 ml of the culture solution, followed by ultrasonic pulverization. The supernatant was centrifuged, The filtrate was filtered under reduced pressure. The filtrate was concentrated under reduced pressure (EYELA, Japan) at 50 ° C until the water remained, followed by solvent fractionation using the same amount of ethyl acetate and butanol. Ethyl acetate, butanol, and water extracts were collected through solvent fraction and concentrated under reduced pressure. Ethyl acetate extract, acetanol, butanol extract methanol and water layer were dissolved in water to investigate nematode activity against root nodule. 96-well tissue culture plates to root-knot nematodes (M. incognita) 2 instar root-knot nematode larvae after tooth shape to have a final concentration of 5% by treatment for each sample to be 100, 1,000 ㎍ / ㎖ (M. incognita) treated larvae Respectively. 5% methanol and 5% acetone were used as untreated control. After 24 hours and 72 hours of treatment, the nematode activity was examined using the equation described above.

Streptomyces net drop system (Streptomyces netropsis ) AN110065 strain was extracted with ethyl acetate and butanol to obtain ethyl acetate, butanol and water extracts. Ethyl acetate extract showed the highest nematode activity at the concentration of 1,000 ㎍ / ㎖ (83.5%) (Fig. 4). Butanol extract did not show nematode activity at the concentration of 1,000 ㎍ / ㎖, and water extract showed a low nematode activity of about 23.4% (FIG. 4).

Example  5. Tomato culture broth To root-knot nematode  Control effect

Streptomyces net drop system (Streptomyces Pot experiments were carried out on tomato root blight nematodes using the culture broth of netopsis AN110065. The root-knot nematode pot experiment was carried out in a glasshouse (25 ± 5 ℃) in 5 research centers of the Korea Research Institute of Chemical Technology. 400 g of sterilized sand was placed in a plastic port measuring 9.5 cm in diameter and seeded on the soil and transplanted in a greenhouse for 2 weeks. As described above, eggs were collected from tomato roots infested with root-knot nematode, and 10,000 eggs were inoculated per each port. Then, AN110065 cultured in GSS medium at 28 ° C and 150 rpm for 10 days with shaking was diluted to 10, 100, and 1,000 times with sterilized water and treated with 4 times of soil circumference around the tomato roots at intervals of one week. As a control, 2,000 times dilution of ai Fosthiazate 5% was treated once, and sterilized water was treated with no treatment. After six weeks of nematode infestation, the roots of the plant were washed thoroughly with tap water, and then Taylor and Sasser (1978, Biology, identification and control of root-knot neamtodes ( Meloidogyne species), Cooperative Publication Department Plant Pathology, The root-knot index was investigated according to the method of North Carolina State University and United State Agency for International Development, North Carolina State University Graphics, Raleigh, USA, 111). The rooting index is applied in 0-5 steps (0 for 0%, 1 for 1-20%, 2 for 21-40%, 3 for 41-60%, 4 for 61-80%, 5 for 81-100%) Respectively.

Statistical analysis was performed by one-way analysis of variance ANOVA using SAS program (SAS Institute, Inc., 1989, Cary, NC) and Duncan's multiple range test ( P = 0.05) Respectively. The control activity against tomato root blight nematodes in the greenhouse was 41, 30, and 22%, respectively, at 10 times, 100 times, and 1,000 times dilution treatment treatments, respectively, showing a concentration-dependent control activity (FIG. Streptomyces net drop system (Streptomyces The net dilution of AN110065 strain was significantly different from that of untreated group, but there was no significant difference between 100 and 1,000 dilutions. The control of the nematodes showed 100% control efficacy, indicating that they completely control the root nematode. The strains of Streptomyces netropsis AN110065 showed poor growth in the 100 - fold and 1,000 - fold dilution treatments at the early stage of growth, but showed vigorous growth at the 10 - fold dilution and no weakness was found.

Korea Biotechnology Research Institute KCTC12490BP 20130923

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY          INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY          Korea Research Institute of Bioscience and Biotechnology <120> Streptomyces netropsis AN110065 strain having nematocidal          activity against root-knot nematode and uses <130> PN15049 <160> 3 <170> KoPatentin <210> 1 <211> 1371 <212> DNA <213> Streptomyces netropsis <400> 1 catgcagtcg aacgatgaag cctttcgggg tggattagtg gcgaacgggt gagtaacacg 60 ttgggcaatc tgcccttcac tctgggacaa gccctggaaa cggggtctaa taccggatac 120 gacctgcctc cgcatggggg tgggtggaaa gctccggcgg tgaaggatga gcccgcggcc 180 tatcagcttg ttggtggggt aatggcctac caaggcgacg acgggtagcc ggcctgagag 240 ggcgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgca caatgggcga aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt 360 cgggttgtaa acctctttca gcagggaaga agcgagagtg acggtacctg agaagaagcg 420 ccggctaact acgtgccagc agccgcggta atacgtaggg cgcaagcgtt gtccggaatt 480 attgggcgta aagagctcgt aggcggcttg ttgcgtcgga tgtgaaagcc cggggcttaa 540 ccccgggtct gcattcgata cgggcaggct agagtgtggt aggggagatc ggaattcctg 600 gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg 660 gccattactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta 720 gtccacgccg taaacgttgg gaactaggtg ttggcgacat tccacgtcgt cggtgccgca 780 gctaacgcat taagttcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat 840 tgacgggggc ccgcacaagc agcggagcat gtggcttaat tcgacgcaac gcgaagaacc 900 ttaccaaggc ttgacatata ccggaaacgg ccagagatgg tcgccccctt gtggtcggta 960 tacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020 cgagcgcaac ccttgttctg tgttgccagc atgcctttcg gggtgatggg gactcacagg 1080 agactgccgg ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat 1140 gtcttgggct gcacacgtgc tacaatggcc ggtacaatga gctgcgatac cgtgaggtgg 1200 agcgaatctc aaaaagccgg tctcagttcg gattggggtc tgcaactcga ccccatgaag 1260 ttggagttgc tagtaatcgc agatcagcat tgctgcggtg aatacgttcc cgggccttgt 1320 acacaccgcc cgtcacgtca cgaaagtcgg taacacccga agccggttcc c 1371 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggttaccttg ttacgactt 19

Claims (9)

Streptomyces netropsis AN110065 strain (KCTC12490BP) with live nematode activity against Meloidogyne incognita . delete delete delete A microorganism preparation for the nematode of Meloidogyne incognita containing the strain, spore, culture or cultured extract of claim 1 as an active ingredient. The microorganism preparation according to claim 5, wherein the microorganism preparation further comprises lime. The microorganism preparation for Meloidogyne incognita . Claim 5 method of controlling the marshmallow Ido Guinea encoding cognitive other steps Melo Ido Guinea encoding cognitive other (Meloidogyne incognita) comprising a handling a microbial agent for a nematocide for the crops, seeds or cultivation of the crop (Meloidogyne incognita). A method for preparing a microorganism preparation for the nematode of Meloidogyne incognita , which comprises culturing the strain of claim 1. 9. The method according to claim 8, further comprising the step of extracting the culture obtained after culturing the strain with ethyl acetate.

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