KR101362211B1 - Photorhabdus temperata subsp. temperata and antimicrobial and insecticidal composition having extracts derived from the same - Google Patents

Abstract

The present invention relates to photorhabdus temperata subsp. temperata and an antimicrobial and insecticidal composition having extracts derived from the same, and more specifically to photorhabdus temperata subsp. temperata having both excellent antimicrobial activity and excellent insecticidal activity and an antimicrobial and insecticidal composition having extracts derived from the same. Photorhabdus temperata subsp. temperata ECOWIN_104 according to the present invention has superior insecticidal activity against crop pests and antibacterial activity against plant pathogens thereby effectively and environmentally friendly controlling and preventing damage caused by crop pests in various crops by using the culture medium or the extracts thereof.

Description

Antimicrobial and insecticidal composition comprising photolabdus temperata tempprata and extracts thereof [Photorhabdus temperata subsp. temperata and antimicrobial and insecticidal composition having extracts derived from the same}

The present invention relates to an antimicrobial and insecticidal composition comprising photolapdus temperata temperata and extracts thereof, and more specifically, to a photolabdus temperata temperata and extracts thereof having both excellent antimicrobial and insecticidal activity. It relates to the antimicrobial and insecticidal composition included.

Insect pathogens derived from entomopathogenic nematodes are known to have immunosuppressive effects, cytotoxic effects, antibiotic secretion and insecticidal activity. To understand whether this ability is derived from a species, it is necessary to understand the life cycle represented by insect pathogens.

Insect pathogens belong to Enterobacteriaceae and have a symbiotic relationship with insect pathogenic nematodes. In other words, these insect pathogens are present in the intestinal worm's bowel. When the target insects appear, infectious nematodes invade into the bloodstream of host crop pests, and intestinal symbiotic bacteria are spewed out into the bloodstream of insects.

Hemolymph, which contains a mixture of vertebrate blood and lymph fluid, is located in the bloodstream of insects. Hemolymph is divided into plasma and blood cells, and has almost similar functions in addition to oxygen carrying ability of vertebrate blood. In particular, hematopoietic cells have cellular immune responses against foreign invaders such as nematodes or bacteria, and are known to be capable of humoral immune responses because plasma has various antibiotic proteins.

This insect immune response consists of the recognition of foreign invasive substances, mediation and immune reaction.

The immediate problem with invading nematodes and symbiotic bacteria released from them is the immune defense mechanisms of insect blood lymphocytes. In general, pathogens exert mechanisms of immune recognition avoidance, inhibition and resistance. However, the precise distinction between them is very difficult, and in fact, these mechanisms appear to be a phenomenon of the immune system that is a combination of each other.

In the case of Xenorhabdus nematophila (Xn), immunosuppression is induced against phospholipase A2 (PLA2). PLA2 inhibition by Xenorabdus nematophila does not lead to an immune response, even though blood cells recognize the invasion of foreign pathogens and do not produce icosanoids.

After suppressing the immune action, genoblastus nematophila directly induces cell death. In particular, lethality of blood cells eventually results in irreversible inhibition of immune responses due to blood cells.

On the other hand, the immunosuppression of insects caused by Xenorabdus nematophila can allow invasion of other pathogens in nature. Since the development of symbiotic nematodes requires the monoxenic condition of symbiotic bacteria alone, it is necessary to secrete antibiotics capable of inhibiting similar bacterial species or even taxonomically very heterogeneous types of bacteria and fungi.

Among the components contained in the secondary metabolites of insect pathogens, it is known that benzelidenaceton, an immunosuppressive substance, and anthraquinon, which have antimicrobial properties, include various toxin proteins.

Although wandering bacteria with pathogenic to insects half of the insect lethal dose (LD50) is a gen drawer Douce (Xenorhabdus) in bacteria of the host one type of insect pathogenic bacteria only to be considered as insect pathogenic bacteria if 10,000 cells or less When inserted with a nematode of the Steinernema genus, it exhibits high pathogenicity to the bee-larged moth larva with a concentration of LD50 <100 cells. However, only symbiotic bacteria without host nematodes, LD50> 5,000 cells, show very low pathogenicity by symbiotic bacteria. Became known.

However, among the symbiotic bacteria of nematodes picture Rob Douce (Photorhabdus) in bacteria do not have to deal with a host symbiotic nematodes are reported to be less than the LD50 for the honeybee larvae of moths, 100 people Debt cells. However, even symbiotic bacteria with high insecticidal activity have a problem in that they are limited to oral toxicity and injection toxicity.

Thus, the present inventors have made a thorough effort to solve the above problems, as a result of selecting a photolabdus temperata temprata strain having both antimicrobial activity and insecticidal activity, that the extract has more excellent antimicrobial activity and pesticidal activity. It confirmed and completed this invention.

An object of the present invention is to provide a microorganism having both antimicrobial activity against phytopathogens and pesticidal activity against crop pests.

Another object of the present invention is to provide an antimicrobial composition and an insecticidal composition comprising an extract of a microorganism having a antimicrobial activity against phytopathogens and pesticidal activity against crop pests, a culture medium thereof and a microbial culture solution.

In order to achieve the above object, the present invention provides Photorhabdus temperata tempprata ECOWIN_104 (Accession Number: KACC 91741P) having both antimicrobial activity and insecticidal activity.

The present invention also provides an antimicrobial composition comprising Photorhabdus temperata tempprata ECOWIN_104 ( Photorhabdus temperata subsp. Temperata ECOWIN_104) (accession number: KACC 91741P) or a culture thereof as an active ingredient.

The present invention also provides a pesticidal composition comprising Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp. Temperata ECOWIN_104) (accession number: KACC 91741P) or a culture thereof as an active ingredient.

The present invention also provides an antimicrobial composition comprising an extract of the Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp. Temperata ECOWIN_104) (accession number: KACC 91741P) as an active ingredient.

The present invention also provides a pesticidal composition comprising an extract of the Photorhabdus temperata tempprata ECOWIN_104 ( Photorhabdus temperata subsp. Temperata ECOWIN_104) (accession number: KACC 91741P) as an active ingredient.

In the present invention, the extract is characterized in that extracted by adding a solvent selected from the group consisting of hexane, ethyl acetate, ethanol and water to the strain culture.

In the present invention, the photolabduus temperata temprata ECOWIN_104 and the antimicrobial composition is Botrytis cinerea, Phytophthora capsici , Colletotrichum acutatum , Fusarium oxysporum , Rhizoctonia solani and It is characterized by having antimicrobial activity against one or more plant pathogens selected from the group consisting of Ryezotonia serealis ( Rhizoctonia cerealis ).

In the present invention, the photolapdus temperata temprata ECOWIN_104 and the insecticidal composition is honey cabbage moth ( Plutella xylostella ), tobacco giant moth ( Spodoptera litura ), moth horned moth ( Alcis angulifera ), moth pattern leaf moth ( Adoxophyes orana), persimmon ipmalyi moth (Ptycholoma lecheana), the night the baby ipmalyi moth (Cydia Kurokoi), peach net moth (Grapholita molesta), is patterned oysters moth (Lyonetia prunifoliella), peach simsik moth (Carposina sasakii), beet armyworm (Spodoptera exigua ), diaphania indica , Cacthalocrocis medinalis , Clamphalocrocis medinalis , Chilo suppressalis and Helicoverpa armigera It is done.

Photolapdus temperata temprata ECOWIN_104 according to the present invention is excellent in both insecticides and pesticides against phytopathogens of crop pests, when using the culture medium or extract thereof, effective and environmentally friendly to damage caused by crop pests in various crops Can be controlled and prevented.

1 is a phylogenetic schematic diagram comparing the 16S rDNA nucleotide sequences of a known strain of Photolabdus genus registered in Photolabdus temperata temprata ECOWIN_104 and NCBI of the present invention.
Figure 2 is a result of comparing the 16S rDNA nucleotide sequence of the known strain of the photolabdus temperata temprata ECOWIN_104 and the genus Photorapdus registered in the NCBI of the present invention.
Figure 3 is a photolabduus temperata tempprata ECOWIN_104 of the present invention and the known strain P. temperata subsp. This is the result of comparing and comparing the base sequences of temperata BE09 NCBI EU930337.
Figure 4 is a photograph of colonies incubated in the NBTA medium of the photolapdus temperata temperata ECOWIN_104 of the present invention.
Figure 5 is a photograph of the liquid culture solution of the Photorabdus temperata tempprata ECOWIN_104 of the present invention.

In the present invention, after separating the insect pathogenic nematode having both antimicrobial activity against phytopathogens and pesticidal activity against crop pests from soil samples, and separating symbiotic bacteria therefrom, antimicrobial activity against phytopathogens and pests against crop pests It was intended to confirm that the strain having all the activities can be selected.

In one embodiment of the present invention, the insecticidal pathogenic nematode having both antimicrobial activity against plant pathogens and pesticidal activity against crop pests is separated from soil samples, and from this, antimicrobial activity against plant pathogens and pesticidal activity against crop pests All strains were selected.

Next, the morphological and biochemical characteristics of the selected strains were observed, and as a result of the identification through the carbon utilization survey and 16S rDNA sequencing, the selected strains were photorhabdus temperata subsp. temperata , which was deposited on September 13, 2012 to the Center for Agricultural Genetic Resources (KACC 91741P).

Accordingly, the present invention relates to Photorhabdus temperata tempprata ECOWIN_104 (accession number: KACC 91741P) having both antimicrobial activity and insecticidal activity in one aspect.

Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession Number: KACC 91741P) is Botrytis cinerea, Phytophthora capsici , Collet tree It has antimicrobial activity against plant pathogens such as Colletotrichum acutatum , Fusarium oxysporum , Rhizoctonia solani , and Rhizoctonia cerealis . do.

The Botrytis cinerea is gray mold, the Phytophthora capsici is pepper blight, and the Colletotrichum acutatum is melon anthrax, the Fusarium oxis Fusarium oxysporum is known to cause wilting disease, and Rhizoctonia solani is known to cause rice leaf blight and Rhizoctonia cerealis to cause rice leaf blight.

Also, the picture Rob Douce tempera other system Prata ECOWIN_104 (Photorhabdus temperata subsp temperata ECOWIN_104. ) ( Accession number: KACC 91741P) is the honey cabbage moth (Plutella xylostella), tobacco giant seminar room (Spodoptera litura), beet armyworm (Spodoptera exigua ), Alcis angulifera , Adoxophyes orana , Persimmon leaf moth ( Ptycholoma lecheana ), Chestnut leaf moth ( Cydia Kurokoi ), Peach net moth ( Grapholita molesta ) Pesticide activity against insect pests such as Lyonetia prunifoliella ), Peach- Carrot Moth ( Carposina sasakii ), Cotton Monarch ( Diaphania indica ), Persimmon Moth ( Cnaphalocrocis medinalis ), Chilo suppressalis and Helicoverpa armigera Characterized in having a.

As confirmed through the experiments, Photorhabdus temperata tempprata ECOWIN_104 ( Photorhabdus temperata subsp. Temperata ECOWIN_104) (Accession No .: KACC 91741P) of the present invention has antimicrobial activity against plant pathogens and pesticidal activity against crop pests. Since both have, it can be used as an antimicrobial composition or a pesticidal composition.

Therefore, in another aspect, the present invention relates to an antimicrobial composition and insecticidal composition comprising Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (accession number: KACC 91741P) or a culture thereof as an active ingredient. .

Meanwhile, in the present invention, an extract having antimicrobial activity against plant pathogens and pesticidal activity against crop pests is isolated from a culture medium of Photorhabdu temperata tempprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession Number: KACC 91741P). In this case, the antimicrobial activity against phytopathogens and pesticidal activity against crop pests could be further improved.

In another embodiment of the present invention, hexane extract, ethyl acetate extract, ethanol extract, and water extract were obtained from the culture solution of Photorhabdus temperata tempprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (accession number: KACC 91741P). It was confirmed that the antimicrobial activity against phytopathogenic bacteria and pesticidal activity against pests of moths crops were obtained.

Therefore, in another aspect, the present invention relates to an antimicrobial composition and an insecticidal composition comprising an extract of Photorhabdus temperata subsp.temperata ECOWIN_104 (Accession No.:KACC 91741P) culture as an active ingredient. will be.

The extract can be extracted by adding a solvent to the photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession Number: KACC 91741P) culture, the culture solution is centrifuged to remove the water and methanol, Polar solvents of C1-C4 lower alcohols such as ethanol, butanol, etc., or nonpolar solvents such as hexane, ethyl acetate, chloroform, dimethyl sulfoxide (DMSO), preferably hexane, ethyl acetate, ethanol and water, are added sequentially By fractionation, it can extract.

As is commonly known, the extract can be used as is, or concentrated and then diluted to the required concentration.

(A) Photorhabdus temperata subsp. Temperata ECOWIN_104 (An accession number: KACC 91741P), (b) a culture thereof or (c) an extract of the antimicrobial and insecticidal active ingredient according to the present invention . Plants that can be applied to the antimicrobial composition and insecticidal composition comprising as an active ingredient are not particularly limited, pepper, cabbage, cabbage, eggplant, persimmon, peach, cucumber, cabbage lettuce (lettuce), strawberries, tomatoes, green onions, In addition to economic crops such as tobacco, rice, chestnuts, etc., they can be treated on plant surfaces that can be a host of pests of moths, such as flowers or special crops, soils on which these plants are grown, and fruit or vegetable surfaces grown, transported or stored. have.

The antimicrobial composition or insecticidal composition of the present invention is an antibacterial and insecticidal active ingredient, Photorhabdus temperata tempprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession No .: KACC 91741P), its culture or dry powder, or extract of the culture medium In addition, it may further include a surfactant, an inorganic salt, a binder, an extender, and the like.

The surfactant is an amphiphilic material having both hydrophilic and lipophilic molecular groups in the molecule, and has excellent characteristics in cleaning, dispersing, emulsifying, solubilizing, wetting, sterilizing, foaming and penetrating properties according to the present invention. Hydration, so that Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp. Temperata ECOWIN_104) (Accession No .: KACC 91741P), its culture or dry powder, or extract of the culture solution in the antimicrobial or insecticidal composition effectively expresses the medicinal effect, It can act to suspend and disperse.

The surfactants include sodium salts of sulfonates such as alkylbenzenesulfonates, alkylnaphthalenesulfonates, dialkylsulfosuccinates, ligninsulfonates, alkylnaphthalenesulfonate formalin condensates, polyoxyalkylenealkylphenylsulfonates, or Sodium salts of sulfates such as calcium salts, alkyl sulfates, polyoxyalkylene alkyl sulfates, polyoxyalkylene alkylphenyl sulfates or sodium salts of succinates such as calcium salts, naphthalenesulfosuccinates, polyoxyalkylene succinates or Anionic surfactants such as calcium salts, ethoxylated alkyl ethers, polyoxyalkylene alkylphenyl polymers, and nonionic surfactants such as multiple alcohols may be used alone or in combination of two or more thereof, all of which are listed by way of example. Surfactants other than these may be used as is conventional in the art. It can be easily understood by those who have knowledge.

The inorganic salts enter the middle of the crop pests together with the photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (accession number: KACC 91741P), its culture solution or dry powder, or the extract of the culture medium. A substance that helps to quickly convert to the form of active toxins or physical damage to the middle barrier to enhance the activity of toxins, as well as to induce physiological changes in the body of crop pests to enhance the effect of toxins As can be easily purchased and used by those skilled in the art.

The binder acts to bind the antimicrobial and insecticidal active ingredients with other extenders, such as water-soluble starch, dextrin, carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, arabic gum or xanthan gum alone or two. What mixed more than 1 type can be used.

The extender is used in conjunction with the surfactant to adsorb and powder the surfactant and, together with the surfactant, antimicrobial and pesticidal active ingredients, to act as a material for forming the particulate surface of the composition, and may contain starch, soybean meal, bran, granular fibres, Yuan, diatomaceous earth, zeolite, bentonite, talc, kaolin, pyrophyllite, white carbon and the like may be used alone or in combination of two or more thereof.

The antimicrobial composition or pesticidal composition according to the present invention may be formulated in the form of granules, powders, liquid hydrating agents, hydrating agents, etc., but is preferably formulated with a hydrating agent. Formulated fungicides or pesticides may be used diluted 500 to 2,000 times, preferably about 1,000 times, in water before use.

When the antimicrobial composition or the pesticidal composition according to the present invention is used for agriculture, the application amount thereof may be usually 0.1 to 100 g per 10 acres, but is not limited thereto. For emulsifiable concentrates, wettable powders, flows and other similar formulations used after dilution with water, their application concentrations are usually in the range of 1 to 100,000 ppm. The amount and concentration of such application may vary depending on the type of formulation, time of application, location and method, type of crop pest or plant pathogen, degree of damage and other factors, and are not limited to the above range and may be increased or decreased.

In addition, the antimicrobial composition or insecticidal composition of the present invention may be used together with or with other insecticides, nematicides, acaricides, fungicides, fungicides, herbicides, plant growth regulators, synergists, fertilizers, soil conditioners, and the like. Separate but can be used simultaneously.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

EXAMPLES Example 1: Selection of Porutabudus templata

1-1: Isolation of Insect Pathogenic Nematodes

The soil samples (Pocheon, Gyeonggi-do) were collected by removing the dry part of the surface and collecting 0.5 ~ 1 kg of soil containing 10 ㎝ depth of water. Respectively. The insect baiting method (Bedding, 1975) was used to isolate insect pathogenic nematodes from the soil samples.

First, 200 g of soil sample was placed in a plastic box, distilled water was sprayed on it, and a trap for searching nematodes was inserted. At this time, one filter paper was covered to prevent contamination of non-pathogenic nematodes when lethal insects were trapped. Then, 100 larvae of honey bee moth larvae were placed on filter paper which was sprayed with sterilized distilled water, and insecticidal effects of larvae were observed at intervals of 24 hours in an incubator where humidity and temperature were kept at 50% and 20 ° C, respectively.

The infected larvae were separated and transferred into test dishes one by one, and the nematodes released from the infected larvae were separated after 2 to 3 days. The separated nematodes were re-inoculated into the larvae of the honeybee larvae and maintained at 20 DEG C for about 24 to 72 hours in the same manner as above.

1-2: Isolation of Symbiotic Bacteria from Insect Pathogenic Nematodes

Symbiotic bacteria were isolated from entomopathogenic nematodes using the following two methods.

The first method is to separate from infectious insect pathogenic nematodes, soak the infectious nematodes in 0.1% sodium hypochlorite (NaClO) solution, surface sterilize the nematodes, and then grind them in sterile Tryptic Soy Broth (TSB) liquid medium. It is a method of crushing or crushing and enriching. The second method is to separate from the honeybee moth larvae infected with the insect pathogenic nematode, inoculate about 10 nematodes per beeworm moth larva and incubate for 12 to 48 hours at 25 ° C. After disinfecting the dead larvae with 95% ethanol, the larvae washed three to four times with sterile distilled water are dissected to cultivate hemolymph (hemolymph) in TSB liquid medium and enrich.

The culture medium enriched for 24 to 48 hours at 28 ° C by the above two methods was diluted in sterile TSB medium, then plated in MacConkey solid medium, and incubated in 28 ° C incubator for 3-5 days.

From the colonies formed on MacConkey solid medium, bright pink or red colonies were selected, and the selected colonies were transferred to NBTA solid medium, bromothymol blue was adsorbed on NBTA medium, and tetrazolium was added. Reducing the chloride (tetrazolium chloride) was selected colonies showing a dark yellow or green color of the colonies. For reference, the composition of the medium used in the examples is shown in Table 1 below.

Media Composition Content (g / L) MacConkey agar Peptone 17 Proteose peptone 3 Lactose 10 Even salts 1.5 Sodium chloride 5 Neutral red 0.03 Crystal violet 0.001 Agar 13.5 Tryptic Soy Broth (TSB) Pancreatic digest of casein 17 Papaic digest of soybean 3 Dextrose 2.5 Sodium chloride 5 Dipotassium phosphate 2.5 NBTA Nutrient agar 20 Bromothymol blue 0.025 2,3,5-triphenyl tetrazolium chloride 4

1-3: Insect Pathogenicity eelworm  Confirmation of Morphological Characteristics of Symbiotic Bacteria

In order to identify the entomopathogenic nematode symbiosis bacteria isolated in 1-2, the morphological characteristics were observed through optical microscopy.

In addition, in order to investigate the biochemical properties, after 24 hours incubation at 28 ℃ in TSA (Tryptic Soy Agar) slope medium, the Gram staining reaction was identified using 3% KOH solution, bromine thiblue and glucose containing After inoculating the strain into the medium, and incubated for 2 days at 30 ℃ covered with liquid paraffin and confirmed the oxygen demand through color development. Indole production was inoculated with 1% TSB (Tryptic Soy Broth) liquid medium, incubated for 2 days at 27 ° C, and 10 drops of Kovacs' reagent were observed to observe the change. Shown in

characteristic
(Characters) Photorhabdus Isolated strain Host
(Host) Heterorhabditis sp. Heterorhabditis sp. Gram stain
(Gram staining) ^{-1) -1)} Oxygen composition
(Oxygen requirements) Facultatively anaerobic Facultatively anaerobic Indole production
(Indol production) d + ¹⁾ shape
(shape) rod rod Cell length
(Cell length) 4.0-10.0 탆 4.5-9.2 탆 Cell width
(Cell width) 0.8-2.0 탆 0.82-1.28 탆 Flat hair type
(Filagella type) Peritrichous Peritrichous motility
(Mortility) Sinusodal Motility Sinusodal Motility Bioluminescence
(Bioluminescence) + ¹⁾ + ¹⁾

¹⁾ +: (Positive reaction), - (Negative reaction), d: (weak reaction)

As shown in Table 2, it was confirmed that the insect pathogenic nematode symbiosis bacteria isolated in 1-2 were strains of the genus Photorapdus as compared with the items described in the Bugis manual.

1-4: Identification

1-4-1: Carbon Source Survey

After confirming that the insect pathogenic nematode symbiosis bacteria isolated at 1-2 were bacteria belonging to the genus Photorapdus, a carbon utilization survey was conducted to identify the species. Bacteria were inoculated and cultured using a microplate of Biolog, USA, and the use of carbon sources was examined. The examined results were compared with known strains of the genus Photorapdus, and the results are shown in Table 3.

Characteristic
(Carbon source) P. luminensis P.temperata P.temperata subsp. temperate Isolated strain Trehalose [+] [+] + + L-fucose d d + + D, L-glycerate [-] + + + Mio-Inositol + [+] d d D, L-lactate [-] - - [-] D-mannitol d [-] - -

+: 90% or more, [+]: 76 to 89%, d: 26 to 75%, [-]: 11 to 25%

From the results of Table 3, it was confirmed that the isolated strain is P. temperata subsp. Temperata of the photolapdus genus.

1-4-2: Sequencing of 16S rDNA

Sequencing of 16S rDNA was performed to more clearly identify the species for the isolate strain.

First, genomic DNA was extracted from the insect pathogenic nematode symbiosis bacteria isolated in 1-2 using genomic DNA extraction kit.

In other words, add the symbiotic bacteria and DNA extract solution to the tube, incubate for 30 minutes in a 60 ℃ incubator (JSR cat.no JSON-150), then heated at 100 ℃ for 10 minutes and cooled for 3 minutes 5-6 times Repeated. Next, after centrifugation for 10 minutes at 13,200rpm, by loading the precipitate in 1% agarose gel, genomic DNA of the insect pathogenic nematode symbiosis bacteria was extracted.

16S rDNA was amplified by polymerase chain reaction (PCR) using extracted genomic DNA as a template.

The reaction solution for PCR was 2.5 μl 10 × EF-Taq buffer, 0.5 μl 10 mM dNTP, 1 μl 10 pmol forward primer (SEQ ID NO: 1), 1 μl 10 pmol reverse primer (SEQ ID NO: 2), 2.5 U 1 μl / μl EP Taq, 1 μl template DNA (template DNA-50-100ng), and 25 μl of distilled water were mixed in each tube.

[SEQ ID NO 1]: 5'-AGAGTTTGATCCTGGCTCAG-3 '

[SEQ ID NO 2]: 5'-GGTTACCTTGTTACGACTT-3 '

Amplification of 16S rDNA was performed by using a PCR apparatus (PTC-100 MJ Research, Minnesota, USA) for 15 minutes at 95 ° C, followed by 20 seconds at 95 ° C, 40 seconds at 60 ° C, and 1 minute at 72 ° C. Each reaction was repeated 30 times for 30 seconds, and finally, the reaction was performed at 72 ° C. for 5 minutes.

Next, the sequence of the PCR amplification product was analyzed using a Sequencer (ABI 3730XL DNA Analyzer). BigDye® Therminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Scoresby, VIC, Australia) was used for sequencing.

Sequencing confirmed that the total number of bases (SEQ ID NO: 3) of 16S rDNA of the insect pathogenic nematode symbiosis bacteria isolated at 1-2 was 1509.

First, a phylogenetic tree was prepared by using neighborjoining methods by comparing 16S rDNA sequences with Photorhabdus type strain, and shown in FIG. 1. As shown in red in Figure 1, it was confirmed that the insect pathogenic nematode symbiosis bacteria isolated from 1-2 is a P.temperata subsp. Temperata strain of the genus photorapdus .

Next, the homology between the 16S rDNA sequences of the photolobus genus strains registered in NCBI and the 16S rDNA sequences of the insect pathogenic nematode symbiosis bacteria isolated from 1-2 was investigated using Blastn of NCBI (see FIG. 2). ), P. temperata subsp. The base sequences of temperata BE09 NCBI EU930337 were compared and shown in FIG. 3.

As shown in Figure 3, 16S rDNA of the insect pathogenic nematode symbiotic bacteria isolated from 1-2 is known strain P. temperata subsp. temperata BE09 NCBI EU930337 was found to have 99.2% homology with 16S rDNA. However, as indicated by the underline, since the sequences are not completely identical, the insect pathogenic nematode symbiosis isolated at 1-2 was finally confirmed to be a new strain belonging to the species.

According to the results of homology using 16S rDNA, the insect pathogenic nematode symbiosis isolated from 1-2 was confirmed to be P.temperata subsp.temperata of the genus Photorapdus. It was named Tatempprata ECOWIN_104 (Photorhabdus temperata subsp. Temperata ECOWIN_104) and deposited on September 13, 2012 at the National Institute of Agricultural Science, Agricultural Genetic Resource Center (Accession Number: KACC 91741P).

Example 2: Confirmation of the antimicrobial activity of photolapdus temperata tempprata ECOWIN_104

Centrifugation of the Photolapus temperata temprata ECOWIN_104 culture medium at 5,000 rpm for 20 minutes, and then hexane, ethyl acetate, ethanol and water were added sequentially to the supernatant, and fractionated. Extracts were obtained respectively.

Each extract was diluted to 500 ppm and plated on a plate medium, followed by Botrytis cinerea (KACC 40573), Phytophthora capsici (obtained from Keimyung University) , Colletotrichum orbiculare (obtained from Keimyung University) , Fusarium oxysporum (Department of Microbiology, Keimyung University), Rhizoctonia solani (Department of Microbiology, Keimyung University) and After inoculating Rhizoctonia cerealis (obtained: Department of Microbiology, Keimyung University), the activity was measured after 12 hours, and the results are shown in Table 4.

The phytopathogens used in this experiment were all grown well in the common potato agar medium (Potato dextrose agar medium: PDA), so they were grown on PDA and tested for the antimicrobial activity of plant extracts. First, PDA was prepared by adding 500 ppm of symbiotic extract, and each pathogen mycelial section taken with a 5mm diameter cork borer was placed on it, and cultured in a 25 ℃ thermostat.

The antimicrobial effect of the extract was analyzed by comparing the mycelial growth of pathogens of the control group (untreated) and the extract treated 4 days after the extract treatment. As a control, PDA medium using pure water was used without extract treatment.

Treatment Inhibition rate (%) Botrytis cinerea Phytophthora capsici Colletotrichum acutatum Hexane extract 22.12 ± 0.3 13.34 ± 0.5 12.13 ± 2.4 Ethyl acetate extract 72.24 ± 3.2 62.13 ± 0.9 60.35 ± 0.4 Ethanol extract 54.35 ± 1.3 32.33 ± 0.1 45.72 ± 1.1 Water extract 12.33 ± 0.7 6.54 ± 0.2 5.44 ± 0.3 Non-treatment - - - Treatment Fusarium
oxysporum Rhizoctonia
solani Rhizoctonia
cerealis Hexane extract 25.43 ± 1.8 15.84 ± 0.7 17.26 ± 1.1 Ethyl acetate extract 62.4 ± 1.2 67.76 ± 1.1 75.63 ± 0.6 Ethanol extract 55.66 ± 2.8 51.66 ± 0.9 54.57 ± 1.4 Water extract 5.66 ± 0.8 6.77 ± 2.1 7.71 ± 1.5 Non-treatment - - -

As shown in Table 4, the solvent extracts of the cultured photolapus temperata temprata ECOWIN_104 were all plant pathogens, Botrytis cinerea, Phytophthora capsici , and choletotricum . Colletotrichum acutatum , Fusarium oxysporum , Rhizoctonia solani and It has an antimicrobial activity against Rhizoctonia cerealis ( Rhizoctonia cerealis ), in particular, it was confirmed that the antibacterial activity of the ethyl acetate extract and ethanol extract.

Example 3: Confirmation of the insecticidal power of photolapdus temperata tempprata ECOWIN_104

The solvent extracts of the Photolapdus temperata tempprata ECOWIN_104 culture broth obtained in Example 2 were prepared by diluting with DMSO 500 times, and the disks of Chinese cabbage leaves were made 10 × 10 Cm to 30 times with 500 times of each formulated formulation. the second was immersed then brought out sufficiently shade over 30 minutes, the Petri dish with the air could work in the upper cabbage moth (diameter:: 10Cm, height 4Cm) (Spodoptora exigua ) and tobacco moth ( Spodoptora) litura ) 3 ~ 4 year old larvae (prepared by Kyungpook Biotechnology Research Institute # 313 Bioavailability Research Institute) were placed 5 each. At this time, the temperature of the kennel was maintained at 25 ± 3 ℃, humidity 40 ~ 60%. And water was sprayed on untreated.

After the treatment, the larvae were touched with a small brush for 3 days at 24 hours intervals, and the body was observed to be dead. The results are shown in Table 5.

Treatment Insecticide rate (%) Tobacco Seminar Room Cabbage moth Hexane extract 34.54 ± 0.4 48.54 ± 0.8 Ethyl acetate extract 73.12 ± 0.3 85.46 ± 2.2 Ethanol extract 62.13 ± 0.3 67.63 ± 0.7 Water extract 53.22 ± 0.5 51.2 ± 0.4 Non-treatment 0 0

As shown in Table 5, the solvent extracts of the Photolapdus temperata tempprata ECOWIN_104 culture medium have insecticidal activity against the larvae of Chinese cabbage moth and tobacco germweed moth, and especially the pesticidal activity of ethyl acetate extract and ethanol extract. It was confirmed that it was excellent.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Agricultural Biotechnology Research Institute KACC91741P 20120913

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Claims (13)

Botrytis cinerea, Phytophthora capsici , Colletotrichum acutatum , Fusarium oxysporum , Rhizoctonia solani ) And Antimicrobial activity against one or more plant pathogens selected from the group consisting of Rhizoctonia cerealis and one or more crop pests selected from the group consisting of Plutella xylostella and Spodoptera litura Photorhabdus temperata temprata ECOWIN_104 (accession number: KACC 91741P) having simultaneously insecticidal activity against.
delete delete Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession No .: KACC 91741P) or its culture medium as an active ingredient, Botrytis cinerea, Phytophthora capsaici ( Phytophthora capsici ), Colletotrichum acutatum , Fusarium oxysporum , Rhizoctonia solani and Antimicrobial composition, characterized in that it has an antimicrobial activity against at least one plant pathogen selected from the group consisting of Rhizoctonia cerealis ( Rhizoctonia cerealis ).
delete Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession No .: KACC 91741P) or its culture medium as an active ingredient, Plutella xylostella and Tobacco moth moth ( Spodoptera litura ) Insecticidal composition, characterized in that it has insecticidal activity against at least one crop pest selected from the group consisting of.
delete Photorhabdus temperata tempprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession No .: KACC 91741P) Contains an extract of the culture medium as an active ingredient, Botrytis cinerea, phytophthora capsaici ( Phytophthora capsici ), Colletotrichum acutatum , Fusarium oxysporum , Rhizoctonia solani and Antimicrobial composition, characterized in that it has an antimicrobial activity against at least one plant pathogen selected from the group consisting of Rhizoctonia cerealis ( Rhizoctonia cerealis ).
The antimicrobial composition of claim 8, wherein the extract is extracted by adding a solvent selected from the group consisting of hexane, ethyl acetate, ethanol, and water to the strain culture medium of claim 1.
delete Photorhabdus temperata temprata ECOWIN_104 ( Photorhabdus temperata subsp.temperata ECOWIN_104) (Accession No .: KACC 91741P) Contains the extract as an active ingredient, Chinese cabbage moth ( Plutella xylostella ) and tobacco moth ( Spodoptera litura ) Insecticidal composition, characterized in that it has insecticidal activity against at least one crop pest selected from the group consisting of.
The insecticidal composition according to claim 11, wherein the extract is extracted by adding a solvent selected from the group consisting of hexane, ethyl acetate, ethanol and water to the strain culture medium of claim 1.
delete

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