KR101820010B1 - Enterobacter sp. HK169 having nematicidal effect and uses thereof - Google Patents

Abstract

The present invention relates to novel Enterobacter sp. HK169 strains having a nematicidal effect, and a nematocide containing a culture thereof as an active ingredient. The Enterobacter sp. HK169 strains of the present invention has a high nematode activity against root-knot nematodes, and a culture medium of the strains has a controlling effect against tomato root nematodes. Therefore, the Enterobacter sp. HK169 strains and the culture thereof can be useful as a plant disease control agent through nematode activity.

Description

The present invention relates to a novel Enterobacter sp. Strain HK169 having a nematicidal activity and a culture thereof as an active ingredient. HK169 having nematicidal effect and uses thereof}

The present invention relates to a novel microorganism preparation containing an Enterobacter strain HK169 strain and a culture thereof as an active ingredient, and more particularly to a microbial agent containing an Enterobacter sp. sp.) strain HK169 or a culture solution of the strain as an active ingredient, and a method for controlling root blight nematodes using the strain.

The loss of crops caused by plant-parasitic soil nematodes is estimated to exceed $ 100 billion annually worldwide. Soil nematode is a nematode that is parasitic to plants and free from soil in animals belonging to linear animals. It is a very small organ of 0.2 ~ 0.5mm size belonging to nematode and it is difficult to visually identify it and it can be observed under a microscope. Most nematode forms are threadlike, thin or arcuate, symmetrical, with no nodules on the body and wrinkles on the body surface. It is a soil nematode parasitic to plants. When it is parasitic on crops, it makes the roots of tomatoes, carrots, sweet potatoes, tobacco, and apple trees crumble and rotten roots such as radishes, burdocks, potatoes, chrysanthemums and peaches. do.

In particular, the root-knot nematode ( Meloidogyne spp . ), Which belongs to the plant-derived soil nematode, is parasitic on plant roots and causes serious damage to important crops. The root-knot nematode penetrates near the root growth point of the crop and grows while nourishing the nutrients in the root of the plant. In this process, the root of the crop turns into hump by the action of the hormone secreted by the nematode. This parasitism infects other pathogenic microorganisms or interferes with root growth and prevents the normal growth of the crops. Finally, in Korea, about 54% of the seedlings in Sungju County, Gyeongbuk Province are infected with root-knot nematodes . Globally, loss of edible crops by soil nematodes is estimated at about 11% per year, and the loss to major economic crops such as major vegetables and fruits is about 14% per year, resulting in more than $ 80 billion loss annually worldwide It was reported to come.

Agriculture is an important nematodes in potato root-knot nematode (Meloidogyne incognita), carrot root-knot nematode (Meloidogyne hapla), Java root-knot nematode (Meloidogyne javanica and peanut nematodes ( Meloidogyne arenaria ) are known, and they mainly damage fruits and vegetables such as melon, cucumber, tomato, strawberry, pepper and ginseng. In particular, Ido Melo to root-knot nematode, which is a problem in the country Magnificent other facilities plantations Guinea Inco (Meloidogyne incognita) and Melo Ido Guinea Arena Ria (Meloidogyne arenaria , and fumigants such as methyl bromide and fosthiazate and carbamates have been mainly used as a method for controlling root-knot nematodes.

However, these chemical precautions cause problems such as risk to human being, residual toxicity and environmental pollution.

Plant pathogenic nematodes are particularly difficult to control because they are covered with thick, impermeable cuticles, or envelopes, and have very little sensory nerve cells. Although many pest control compounds can act as neurotoxic agents, the few nerve cells exposed in phytopathogenic nematodes reduce the effective target area of the nematode compound, thus requiring the continuous development of nematode compounds with extremely high neurotoxicity. In addition, since plant pathogenic soil nematodes are mainly found in soil or plant roots, it is relatively difficult for the nematode to be exposed to the control agent, and there is a possibility that the toxic compound seeps into the ground, , Use of neurotoxin based nematicide may contaminate both soil and surface water.

It is also known that methyl bromide, which is one of the most widely used methods of disinfection using fumigants, has a high toxicity to humans and has harmful effects on the stratospheric ozone layer. Recently, the Montreal Protocol , And the disinfection of soil fumigation has the risk of killing the organisms and beneficial microorganisms in the soil indiscriminately.

Also, carbamates, known as the most effective non-fumigant nematicides, are known for their high toxicity, and since August 2010, the carbamate production company, Bayer, We agreed to cancel all production registrations for citrus fruit (citrus), and the carbamate produced by the company will be fully terminated at the end of August 2018.

In addition, there is a disadvantage in that the physical control method is cumbersome and costly to control every three years.

In view of these limitations, there is a need for the use of environmentally safe, eco-friendly methods.

There is a method of using microorganisms as an environment-friendly control method for controlling plant parasitic nematodes. It has been reported that microorganisms have various nematicidal activities by producing various secondary metabolites while directly growing nematodes (Zhao et al., 2014). Therefore, there is a need to develop a strain capable of controlling root nodule nematodes by selecting strains having excellent nematicidal activity.

Korean Patent Laid-Open Publication No. 2009-0111514 discloses a strain of Penny Bacillus polymyxa GBR508 having antimicrobial activity, nematode and plant growth promoting effect, a complex disease control method of plants using same, and a plant growth promotion method, 2015-00159360 discloses a new microorganism strain and a microorganism preparation for environmentally friendly fertilizer. However, the Enterobacter strain having the nematode activity against the root-knot nematode of the present invention and its use have never been disclosed.

Under these circumstances, the present inventors conducted research to find a biomaterial having useful utility as a commercial pesticide against root-knot nematode, and found that an enterobacter strain HK169 isolated from tomato roots exhibited a high nematode potency against high root-knot nematodes And confirming that the culture solution of the above strain has a controlling effect against tomato root blight nematode, thereby confirming that the HK169 strain can be usefully used as a composition for nematode control through the nematode activity, thereby completing the present invention Respectively.

It is an object of the present invention to provide a plant disease controlling agent containing a novel enterobacterium strain HK169 having a carnivorous effect and a culture thereof as an active ingredient.

In order to achieve the above object, the present invention provides a novel genus Enterobacter (deposited as Enterobacter KACC81020BP sp.) strain HK169.

In addition, the present invention provides a composition for controlling nematode comprising the strain or a culture thereof as an active ingredient.

Further, the present invention provides a method for controlling nematode comprising the step of treating the nematode controlling composition to at least one selected from the group consisting of crops, seeds of crops or cultivated areas.

In addition, the present invention provides a method of culturing a strain of Enterobacter HK169 comprising the steps of:

1) Inoculating the strain of claim 1 into a sterile medium; And

2) Shake-culturing the strain of step 1) above.

The HK169 strain of the genus Enterobacteria of the present invention has a high nematode activity against root-knot nematodes, and the culture solution of this strain has a controlling effect against tomato root nematodes. Therefore, the strain HK169 and its culture are used as a plant disease control agent Can be usefully used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a colony shape (A) and a scanning electron microscopic photograph (B) of strain HK169 in Enterobacter.
2 is a diagram showing a phylogenetic tree based on the nucleotide sequence (A) and the gyrB nucleotide sequence (B) of 16S rRNA of the HK169 strain of Enterobacter.
FIG. 3 is a graph showing in vivo nematode activity against root-knot nematodes in the culture medium of strain HK169 in Enterobacter.
FIG. 4 is a graph showing the effect of root-knot nematode control on the culture medium of strain HK169 in Enterobacter.
Fig. 5 is a photograph showing a control effect of the culture medium of HK169 strain of Enterobacter in culture. Fig.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention provides a novel genus Enterobacter (deposited as Enterobacter KACC81020BP sp.) strain HK169.

Wherein the strain has a base sequence of 16s rRNA of SEQ ID NO: 5 or a base sequence of gyrB gene of SEQ ID NO: 6.

Further, the strain is characterized by having a carnivorous effect.

In the specific examples and experimental examples of the present invention, it was confirmed that a new strain of Enterobacter HK169 was isolated from tomato roots to form white colonies and spores of 0.4 to 1.2 탆 in size on the medium (see Fig. 1) (FIG. 2), it was confirmed that HK169 strain culture broth or culture filtrate showed high nematode activity and control activity against the second instar tomato root blight nematode (FIG. 3 Reference)

In addition, the present invention provides a composition for controlling nematode comprising the strain or a culture thereof as an active ingredient.

In the nematode, the root nematode is selected from the group consisting of Meloidogyne incognita , Meloidogyne hapla , Meloidogyne nematode, javanica ), peanut nematode ( Meloidogyne arenaria ) or tomato root nematode ( Meloidogyne incognita , but is not limited thereto.

Further, the present invention provides a method for controlling nematode comprising the step of treating the nematode controlling composition as described above to at least one selected from the group consisting of crops, seeds of crops or cultivated areas.

In the specific experimental examples of the present invention, when the culture broth of HK169 strain was treated with tomato root-nematode-infected tomato, the root-knot nematode of the tomato root was significantly reduced, Reference).

Therefore, HK169 strain and its culture can be useful as a plant disease control agent through the activity of nematode.

In addition, the present invention provides a culture method of strain HK169 in Enterobacter sp. Comprising the steps of:

1) Inoculating the strain of claim 1 into a sterile medium; And

2) Shake-culturing the strain of step 1) above.

Genus Enterobacter according to the invention (Enterobacter sp.) The strain HK169 is a naturally occurring microorganism in Korea and is characterized by having nematode activity against root-knot nematode.

In more detail, while conducting research to find biomaterials with utility value as pesticides, bacteria were isolated from the roots of tomatoes cultivated for root - knot nematode propagation in the greenhouse of the Korea Research Institute of Chemical Technology. The culture broth of the isolated bacteria had the effect of promoting nematode activity and plant growth. Thus, the present inventors identified the strain as HK169 and identified it as Enterobacter sp. Enterobacter sp. HK169 strain (hereinafter referred to as HK169 strain) was deposited with the International Depository Agency (Rural Development Administration, Agricultural Genetic Resources Center) on May 02, 2016 (Accession No. KACC 81020 BP).

The strain HK169 grows well in a normal medium for bacterial growth such as tryptic soy agar medium, nutrient agar medium and Luria-Bertani agar, Preferably at a temperature of 25 to 30, and an optimum growth temperature of about 30.

The strain HK169 showed concentration-dependent nematicide activity in greenhouse experiments as well as in laboratory conditions when cultured at various concentrations in the root-knot nematode Meloidogyne incognita .

The root-knot nematode according to a preferred embodiment of the present invention may be selected from the group consisting of Meloidogyne incognita , Meloidogyne hapla ), Java rootstock ( Meloidogyne javanica ), Peanut root nematode ( Meloidogyne arenaria ) and tomato root nodule ( Meloidogyne incognita , but the present invention is not limited thereto, and the crop may be preferably tomato, but is not limited thereto.

Since strain HK169 does not show any pathogenicity against crops such as rice, tomato, wheat, cucumber, barley, and red pepper, it can be used as a biological control agent and has an effect of promoting the growth of crops in soil treatment. Accordingly, a microorganism preparation for promoting plant growth and a biological fertilizer containing the strain or the culture liquid of the present invention as an active ingredient are provided. The microorganism preparation of the present invention and the plant to which the biological fertilizer can be applied are not particularly limited, but preferably tomato.

The root-knot nematode can be controlled by treating the environment containing the plant (for example, soil, water) with a composition comprising the strain HK169 itself, the culture thereof or the extract of the culture according to the present invention. If necessary, it can be mixed with a carrier commonly used in the field of crop protection agents and formulated into powders, pellets, granules or solutions to be used as a composition for controlling nematode diseases. As the carrier, water, white carbon, kaolin or the like may be used, and the culture may be a weak culture or a solid culture.

The composition of the present invention may be formulated into powder, pellet, granule, microcapsule or the like so as to contain the culture medium of the cultured cells, lyophilized cells and starch, crude protein and rock powder, and the like. The composition can be obtained by adding other components which are easy to apply according to the target nematodes and crops such as surface activators, inert carriers, preservatives, humectants, feed accelerators, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectants, can do. The compositions of the present invention may be in a form or concentrate or a primary composition suitable for direct application by dilution with an appropriate amount of water or other diluent prior to application. Lignocyclic concentration can be variously applied depending on the nature of the specific formulation, the type of the applied plant, the soil and the climate in the cultivation area, and the like, especially concentrated or direct use.

In the present invention, when a preparation is manufactured using the strain Enterobacter HK169 and its culture, various adjuvants may be used depending on the case. Examples of the adjuvants which can be used herein include surfactants, binders, vegetable oils, mineral oils, anti-settling agents, thickening agents, antifoaming agents, cryoprotectants, preservatives and the like, as long as they are used in the technical field.

The preparation of such a preparation can be carried out according to a conventional method in the field, and the preparation prepared by this method is, for example, an emulsifiable concentrate, a dust, a wettable powder, a soluble concentrate, a granule, A suspension in the form of a thickened composition in which the active ingredient is precipitated as solid particles in water, an oil-based suspension concentrate or an aqueous suspension concentrate. Preferred among these agents are preparations in the form of a concentrated composition, oil-based suspension concentrates or aqueous suspension concentrates in which the active ingredient is precipitated as solid particles in water when diluted with water.

The carrier is divided into a solid carrier and a liquid carrier. The solid carrier may be, for example, animal or vegetable powder such as starch, saccharides, lactose, cellulose powder, cyclodextrin, activated carbon, soybean powder, wheat flour or milk powder; Or mineral powders, for example talc, aged soil, bentonite, organic bentonite, calcium carbonate, calcium sulfate, sodium bicarbonate, zeolite, diatomaceous earth, white carbon, clay, alumina or silica. The liquid carrier may be, for example, water; Alcohols such as ethyl alcohol or ethylene glycol; Ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or isophorone; Ethers such as dioxane or tetrahydrofuran; Aliphatic hydrocarbons such as kerosene, petroleum or liquid paraffin; Vegetable oils such as corn oil, soybean oil, linseed oil, sunflower oil, cottonseed oil, rapeseed oil, esterified rapeseed oil such as Phase II, trade name (manufactured by Loveland Industries, Methylated rapeseed oil, olive oil, castor oil, palm oil or avocado oil; Animal oil, such as tallow or whale oil; Mineral oil such as machine oil, heavy oil, silicone oil, naphthene solvent, methylnaphthalene or 1-phenyl-1-xylylethane; Aromatic hydrocarbons such as xylene, trimethylbenzene, tetramethylbenzene, cyclohexane or solvent naphtha; Examples of the solvent include a hydrophilic organic solvent such as N, N-dimethylformamide, N, N-dimethylacetamide, dimethylsulfoxide, cyclohexanone,? -Butyrolactone, - pyrrolidone, N-dodecyl-2-pyrrolidone, tetrahydroxyfurfuryl alcohol or propylene glycol monomethyl ether; Esters such as ethyl acetate or glycerol esters of fatty acids; Or nitrile, such as acetonitrile.

When the nematocide composition of the present invention is formulated as a concentrated composition in which the active ingredient is precipitated as solid particles in water when diluted with water, a hydrophilic organic solvent is used as a carrier. The hydrophilic organic solvent is preferably N, N-dimethylformamide, dimethylsulfoxide, cyclohexanone, gamma -butyrolactone or N-methyl-2-pyrrolidone. The appropriate mixing ratio of the anthranilamide compound or its salt and the hydrophilic organic solvent is usually 1: 500 to 2: 1, preferably 1:50 to 1: 1, in terms of weight ratio.

When the nematocyst composition of the present invention is formulated in the form of an oil-based suspension concentrate, at least one oil-based liquid carrier selected from the group consisting of vegetable oil, mineral oil and animal oil is used. The proper mixing ratio of the anthranylamide compound or its salt and the oil-based liquid carrier is usually 1: 500 to 1: 4, preferably 1:50 to 1:10 by weight.

When the nematocidal composition of the present invention is formulated in the form of an aqueous suspension concentrate, a suitable mixing ratio of anthranilamide compound or its salt and water is usually 1: 500 to 2: 1, preferably 1: 50 to 1: : 1.

In the present invention, other pesticides such as an insecticide, a biting agent, a nematicide, a soil insecticide or a bactericide may be mixed and used in combination. In this case, a more excellent effect may be exhibited.

Among the above pesticides, active compounds such as insecticides, acaricides, nematicides and soil insecticides include, for example, the following (generic names; some of them being applied): propenophos, But are not limited to, dichlorvos, fenamiphos, fenitrothion, EPN, diazinon, chlorpyrifos-methyl, acephate, prothiofos, fosthiazate, phosphocarb, cadusafos, dislufoton, chlorpyrifos, demeton-S methionine, dimethoate, methamidophos, imicyafos, isoxathion, isofenphos, ethion, etrimfos, ), Quinalphos, dimethylvinphos, sulprofos, thiometh (th iometone, vamidothion, pyraclofos, pyridaphenthion, pirimiphosmethyl propaphos, phosalone, formothion (see, for example, formothion, malathion, tetrachlovinphos, chlorfenvinphos, cyanophos, trichlorfon, methidathion, phenthoate, , ESP, azinphos-methyl, fenthion, heptenophos, methoxychlor, paration, monocrotophos, parathion-methyl, organophosphate compounds such as -methyl, terbufos, phospamidon, phosmet, phorate;

But are not limited to, carbaryl, propoxur, aldicarb, carbofuran, thiodicarb, methomyl, oxamyl, ethiophenecarb, ethiofencarb, pirimicarb,

But are not limited to, fenobucarb, carbosulfan, benfuracarb, bendiocarb, furathiocab, isoprocarb, (metolcarb), xylylcarb

carbamate compounds such as xylylcarb, XMC, fenothiocarb;

Neris toxin derivatives such as cartap, thiocyclam, bensultap, thiosultap-sodium; dicofol, tetradifon, endosulfan ( organic chlorine compounds such as endosulfan, dienochlor, and dieldrin;

Organometallic compounds such as fenbutatin oxide, cyhexatin;

But are not limited to, fenvalerate, permethrin, cypermethrin, deltamethrin, cyhalothrin, tefluthrin, ethofenprox, Flufenprox, fenpropathrin, bifenthrin, imidate, cyfluthrin, flucythrinate, fluvalinate, and the like. Cycloprothrin, lambda-cyhalothrin, pyrethrins, esfenvalerate, tetramethrin, resmethrin, pro-estradiol, Such as protrifenbute, zeta-cypermethrin, acrinathrin, alpha-cypermethrin, allethrin, gamma-cyhalothrin gamma-cyhalothrin, theta-cypermethrin, tau-fluvalinate, But are not limited to, tralomethrin, profluthrin, beta-cypermethrin, beta-cyfluthrin, metofluthrin, phenothrin, Pyrethroid compounds such as < RTI ID = 0.0 >

The compounds of the present invention may be used in combination with other drugs such as diflubenzuron, chlorfluazuron, teflubenzuron, flufenoxuron, lufenuron, novaluron, triflumuron, Benzoyl urea compounds such as hexaflumuron, bistrifluron, noviflumuron, fluazuron;

A larval hormone-like compound such as methoprene, pyriproxyfen, fenoxycarb, and diophenolan (diofenolan);

Pyridazinone compounds such as pyridaben;

But are not limited to, fenpyroximate, fipronil, tebufenpyrad, ethiprole, tolfenpyrad, acetoprole, pyrafluprole, Pyrazole compounds such as pyriprole;

But are not limited to, imidacloprid, nitenpyram, acetamiprid, thiacloprid, thiamethoxam, clothianidin, nidinotefuran, Neonitinoids such as dinotefuran;

Hydrazine compounds such as tebufenozide, methoxyfenozide, chromafenozide, halofenozide and the like;

Pyridine compounds such as pyridaryl, flonicamid and the like;

Tetronic acid compounds such as spirodiclofen and the like;

Strobilurin compounds such as fluacrypyrin and the like;

Pyridineamine compounds such as flufenerim and the like;

Dinitro compounds; Organic sulfur compounds; Urea compounds; Triazine compounds; Hydrazone compounds; As other compounds, there may be mentioned buprofezin, hexythiazox, amitraz, chlordimeform, silafluofen, triazamate, Pymetrozine, pyrimidifen, chlorfenapyr, indoxacarb, acequinocyl, etoxazole, cyromazine, 1,3, - 1,3-dichloropropene, diafenthiuron, benclothiaz, flufenerim, bifenazate, spirotetramat, propargyl, But are not limited to, propargite, verbutin, spiromesifen, thiazolylcinnanonitrile, amidoflumet, flubendiamide, clofentezine, Metaflumizone, chlorantraniliprole < RTI ID = 0.0 > ), Cyflumetofen, cyenopyrafen, pyrifluquinazone, fenazaquin, pyridaben, chlorobenzoate, sulphuramide, sulfenamide, sulfuroramid, hydramethylnon, metaldehyde, ryanodine, HGW 86, IKA-2000. Bacillus thuringiens aizawai, Bacillus thuringiens kurstaki, Bacillus thuringiens israelensis, Bacillus thuringiens japonensis, Bacillus thuringiens japonensis, Bacillus thuringiens japonensis, Bacillus thuringiens japonensis, , Crystal protein toxins produced by Bacillus thuringiens tenebrionis or Bacillus thuringiens, microbial pesticides such as insect hospital viruses, insect pathogenic fungi, nematode pathogenic fungi, avermectin ), Milbemectin, milbemycin, spinosad, emamectin-benzoate, ivermectin, lepimectin, spinetoram, , Antibiotics such as abamectin, emamectin; Natural products such as azadirachtin, rotenone; And repellents such as deet.

Examples of the fungicidally active compounds in the above other pesticides include the following (in general, some of them are in application or in the test code of the Food and Drug Safety Evaluation Institute of Japan): mepanipyrim, Anilinopyrimidine compounds such as pyrimethanil, cyprodinil;

Pyridine amine compounds such as fluazinam;

But are not limited to, triadimefon, bitertanol, triflumizole, etaconazole, propiconazole, penconazole, flusilazole, But are not limited to, myclobutanil, cyproconazole, tebuconazole, hexaconazole, furconazole-cis, prochloraz, metconazole, metconazole, epoxiconazole, tetraconazole, oxpoconazole fumarate, sipconazole, prothioconazole, triadimenol, triethanolamine, The compounds of the present invention may be used in combination with other drugs such as flutriafol, difenoconazole, fluquinconazole, fenbuconazole, bromuconazole, diniconazole, tricyclazole, Probenazole, simeconazole, Azole compounds such as pefurazoate, ipconazole, and imibenconazole;

Quinoxaline compounds such as quinomethionate;

Dithiocarbamates such as maneb, zineb, mancozeb, polycarbamate, metiram, propineb, thiram, and the like, compound;

Organic chlorine compounds such as fthalide, chlorothalonil, quintozene;

Imidazole compounds such as benomyl, thiophanate-methyl, carbendazim, thiabendazole, fuberiazole, cyazofamid;

Cyanoacetamide compounds such as cymoxanil;

But are not limited to, metalaxyl, metalaxyl-M, mefenoxam, oxadixyl, ofurace, benalaxyl, benalaxyl-M, Phenylamide compounds such as kiralaxyl, chiralaxyl, furalaxyl, cyprofuram;

A sulfamic acid compound such as dichlofluanid;

Copper compounds such as cupric hydroxide, oxine copper;

Isoxazole compounds such as hymexazol;

Fosetyl-Al, tolcofos-methyl, S-benzyl O, O-diisopropylphosphorothioate, O-ethyl, S, S-diphenylphosphorothioate , Organic phosphorus compounds such as aluminum ethylhydrogenphosphonate, edifenphos, iprobenfos;

N-halogenothioalkyl compounds such as captan, captafol, folpet;

Dicarboxyimide compounds such as procymidone, iprodione, vinclozolin;

Benzanilide compounds such as flutolanil, mepronil, zoxamide, and tiadinil;

But are not limited to, carboxin, oxycarboxin, thifluzamide, penthiopyrad, boscalid, bixafen, fluopyram, isothianil ( isotianil);

Piperazine compounds such as triforine;

Pyridine compounds such as pyrifenox;

Carbinol compounds such as fenarimol, flutriafol;

Piperidine compounds such as fenpropidine;

Morpholine compounds such as fenpropimorph, spiroxamine, tridemorph;

Organic tin compounds such as fentin hydroxide, fentin acetate;

Urea compounds such as pencycuron;

Cinnamic acid compounds such as dimethomorph, flumorph;

Phenylcarbamate compounds such as diethofencarb:

Cyanopyrrole compounds such as fludioxonil, fenpiclonil;

The use of azoxystrobin, kresoxim-methyl, metominofen, trifloxystrobin, picoxystrobin, oryzastrobin, demystistrobin (see, for example, strobilurin compounds such as dimoxystrobin, pyraclostrobin, fluoxastrobin, fluacrypyrim;

Oxazolidinone compounds such as famoxadone;

Thiazolecarboxamide compounds such as ethaboxam;

Silylamide compounds such as silthiopham;

Amino acid amide carbamate compounds such as iprovalicarb, benthiavalicarb-isopropyl, valiphenal;

Imidazolidine compounds such as fenamidone;

Hydroxyanilide compounds such as fenhexamid;

Benzenesulfonamide compounds such as flusulfamid;

Oxime ether compounds such as cyflufenamid;

Phenoxyamide compounds such as fenoxanil;

Anthraquinone compounds;

Crotonic acid compounds;

Antibiotics such as validamycin, kasugamycin, and polyoxins;

Guanidine compounds such as iminoctadine; And

As other compounds, there may be mentioned pyribencarb, isoprothiolane, pyroquilon, diclomezine, quinoxyfen, propamocarb hydrochloride, , Chloropicrin, dazomet, metam-sodium, nicobifen, metrafenone, UBF-307, diclocymet, proquinazide ( proquinazid, amisulbrom (alias amibromdol), mandipropamid, fluopicolide, carpropamid, meptyldinocap, and the like. BCF051, BCM061, BCM062, AF-0201.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Isolation of H169 Strain in Enterobacter from Tomato Roots

The endophytic bacteria were isolated from the roots of tomatoes grown for root - knot nematode propagation in the greenhouse of the Korea Research Institute of Chemical Technology.

Specifically, the tomato roots were washed with water, cut into 1 cm size, placed in a grinder, water was poured to the extent that the tomato roots were submerged, and then pulverized for 60 seconds. The pulverized samples were sifted through a 25 μm sieve, and 0.1 ml of the suspension was applied to a Luria-Bertani agar (LB: 10% NaCl, 5% yeast extract, 10% trypton) Lt; / RTI &gt; The isolated bacteria obtained a single colony through three subcultures and named HK169 strain. The HK169 strain was shake-cultured in a 500 ml Erlenmeyer flask containing 100 ml of sterilized LB medium at 150 rpm, 30 for 3 days.

< Experimental Example  1> Identification of strain HK169

The HK169 strain isolated in Example 1 above was identified as follows through morphological and gene base sequences after confirming the control activity against tomato root roe nematode in <Experimental Example 2>.

<1-1> Morphological characteristics

The morphological characteristics of HK169 strain were observed.

Specifically, the isolated HK169 strain was shake cultured in an LB agar medium for 3 days in an incubator at 30. The spore morphology and size of the strain were investigated using a scanning electron microscope (SEM, JSM-6700F, JEOL Ltd., Japan). The HK169 strain was immobilized with glutaraldehyde solution (0.2 M sodium cacodylate buffer, 20% paraformaldehyde, 8% glutaraldehyde) and then washed with 0.05 M sodium cacodylate buffer. Then, osmium tetroxide (2%) and cacodylate buffer (0.1 M) were mixed at a ratio of 1: 1 and then dehydrated using ethanol. The dehydrated cells were immersed in 1,1,1,3,3,3-hexamethyldisilazane, evaporated in a hood for one day, And observed with an electron microscope.

As a result, as shown in Fig. 1, the strain HK169 formed a white colony on the medium (Fig. 1 A), and it was confirmed that spores having a size of 0.4-1.2 탆 were formed in a liver-like shape (Fig. 1B) .

<1-2> Sequence analysis of strain HK169

Molecular phylogenetic analysis of the strain HK169 was carried out by 16S rRNA and gyrB sequencing.

Specifically, the strain was shake-cultured in 5 ml LB liquid medium at 30, 150 rpm for 3 days. The culture was centrifuged at 4, 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). For polymerase chain reaction (PCR), the 16S rRNA gene was amplified using a universal primer set [27F; 5'-AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 1), 1492R; 5'-TACGGYTACCTTGTTACGACTT-3 '(SEQ ID NO: 2)] and the gyrB gene was amplified using primers set [gyr-320; 5'-TAARTTYGAYGAYAACTCYTAYAAAGT-3 '(SEQ ID NO: 3), rgyr-1260; 5'-CMCCYTCCACCARGTAMAGTTC-3 '(SEQ ID NO: 4)] was used. Two nucleotide sequences were analyzed using the NCBI BLAST program. As a result, strain HK169 was identified as an Enterobacter sp. Strain.

Sequence alignment was performed with the CLUSTAL W program to compare the 16S rRNA and gyrB nucleotide sequences of the HK169 strain, which was classified as genus Enterobacter, with the species in the enterobacter and other related taxa. The similarity of 16S rRNA and gyrB was calculated using the MEGA 4.0 program based on the neuronal binding method (Saitou and Nei, 1987). The molecular schematics were constructed using the Neighbor-joining method, which is one of the distance matrix methods, and the bootstrap analysis was performed using the algorithm of the MEGA program.

As a result, the phylogenetic tree as shown in FIG. 2 was obtained. The HK169 strain was systematically located in the genus Enterobacter, and bootstrap values supported this. In addition, 16S rRNA in the HK169 strain Enterobacter genus (Enterobacter sp MK33:. 99.79% ) and Enterobacter strains Aspergillus debris Ke (Enterbacter asubiae : 99.59 %) (FIG. 2A). On the other hand, gyrB gene of HK169 strain Enterobacter claw acetate (Enterobacter cloacae: 98.14%) and Enterobacter asbestos debris Ke: exhibited a high degree of similarity with (Enterbacter asburiae 97.90%) (B in Fig. 2). The strain thus isolated was transformed into Enterobacter sp.) strain HK169, deposited with the International Depositary on April 29, 2016 and assigned the deposit number (KACC 81020BP).

< Experimental Example  2> Root-knot nematode Second generation  About larva Nematode  activation

The nematode activity of the second instar larva of tomato root roots was investigated using the culture medium of HK169 strain isolated in Example 1 above.

Specifically, the root-knot nematode used in the experiment was grown in a greenhouse (25 ± 5) using tomatoes of 'Seokwang' (Monsanto Korea) variety, which was isolated and identified by Hwang et al. (2014). 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. In order to isolate the root nematode larvae, tomato root roots infested with root nematode were washed with water, cut into 1 cm size and placed in a pulverizer, and 0.5% sodium hypochlorite solution was poured into the tomato roots to submerge and then pulverized for 60 seconds. The pulverized samples were sieved using a 65 μm sieve and eggs were collected in a 25 μm sieve. The nematode eggs collected in a 25 μm sieve were washed three times with sterilized water to remove the remaining sodium hypochlorite solution, and nematode larvae were obtained.

The suspension of the nematode (J2 larvae) (about 70/100 μl) was added to each well of a 96-well tissue culture plate and treated with 1.25, 2, 5, and 10% of the HK169 strain culture solution so that the final volume per well was 100 μl Respectively. Abamectin was used at the concentration of 1 ㎍ / ㎖ as a control, and the methanol solution was treated at 1% level in the untreated control. After the sample was treated, the 96-well microplate was agitated for 30 seconds to uniformly mix the samples. To prevent oxygen deficiency and drying of the nematode, the plate was placed in a plastic container with a relative humidity of 100% and stored in a 25-27 incubator. The experiment was carried out three times and 48 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), which was determined by using the following Abbott's formula:

(%) = [Mortality rate of treatment - mortality rate of treatmentlessness] /

(100 - mortality rate in untreated)] × 100.

As a result, as shown in FIG. 3, after culturing for 48 hours after the treatment with HK169 strain, the activity was 71.3% at 10% treatment, 89.9% at 5% treatment, 50.7 % Activity (Fig. 3). In addition, the activity was 98.2% at 10% treatment after 72 hours of sample treatment, and 78.1% at 5% treatment, showing higher control activity over time (FIG. 3).

In addition, the results of the experiment using culture broth of HK169 strain were similar to those of the cultured broth. The above results suggest that the killing activity of HK169 strain is due to the nematocide active substance in the culture filtrate or that the bacteria themselves exhibit live nematode activity against the nematode.

< Experimental Example  3> Enterobacteriaceae ( Enterobacter sp ). Control Effect of Culture of HK169 on Tomato Root-knot Nematode

The following in vivo experiment was carried out to confirm the effect of the culture solution of HK169 strain of Enterobacter sp. In controlling tomato root blight nematode.

The root nodule nipple port experiment was carried out in a glasshouse (25 ± 5) in 5 research centers of 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 in a horticultural soil (Bupunsaci) and transferred to a greenhouse grown in a greenhouse for 2 weeks to a tomato (Monsanto Korea). In order to capture root nematode from tomato root infested with root - knot nematode, roots of tomato nematode were strongly harvested and root - knot nematodes were isolated from root - knuckles and ovary. For the isolation of root nodule nematodes, tomato root roots infested with root nematode were washed with water, cut into 1 cm size and placed in a pulverizer, and a 0.5% sodium hypochlorite solution was poured into the tomato roots so as to be submerged, followed by pulverization for 60 seconds. The pulverized samples were sieved using a 65 μm sieve and eggs were collected in a 25 μm sieve. The nematode eggs collected in a 25 μm sieve were washed three times with sterilized water to remove the remaining sodium hypochlorite solution, and nematode eggs were obtained.

The obtained root nodule nematode was inoculated 10,000 times per each port, and then the stock solution and the 1/3, 1/20-fold dilution solution which were cultured in LB medium at 30 and 150 rpm for 3 days with shaking were treated with soil cultivation twice at one week intervals . A 2,000-fold dilution of a.i. fosthiazate 5% was treated once with the control, and sterilized water was treated with an untreated control. Five plants were used per experiment and the experiment was repeated three times. After 6 weeks of nematode instillation, the roots of the plant were washed thoroughly with tap water and then the root-knot index was investigated (Taylor and Sasser, 1978). The rooting index was examined by applying 0 to 4 steps (0 for 0%, 1 for 1 to 25%, 2 for 25 to 50%, 3 for 51 to 75%, 4 for 76 to 100%).

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.

As a result, as shown in FIGS. 4 and 5, the control activity against tomato root nodule nematodes in the greenhouse was not significantly different from that of the untreated control in 1/20 dilution of HK169 culture medium, And 60% and 25%, respectively. These results showed that the concentration-dependent inhibitory activity was observed. The control effect was similar to that of the nematode used as control in the culture broth treatment, and thus it can be used as a microbial pesticide capable of controlling root- (Figs. 4 and 5).

Rural Development Administration Center for Agricultural Genetic Resources KACC81020BP 20160429

Claims (7)

delete delete delete A composition for controlling tomato root nodule ( Meloidogyne incognita ) comprising a culture of a novel Enterobacter sp. HK169 strain deposited with KACC81020BP, which has a nematicidal effect against a tomato root nematode ( Meloidogyne incognita ), as an active ingredient, As a result,
The culture medium is a novel Enterobacter genus (Enterobacter sp.) HK169 strain the LB (Luria Bertani) was shaken for 3 days in a medium tomato, it characterized in that a culture broth root-knot nematode (Meloidogyne incognita) controlling composition for deposit to KACC81020BP.
delete The tomato root-knot nematode ( Meloidogyne incognita ) controlling at least one selected from the group consisting of crops, seeds of crops or cultivated areas.
delete

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