KR101971576B1 - A composition for controling plant disease and promoting plant growth comprising mariniflexile rhizosphaerae trm1-10 - Google Patents

Abstract

The present invention relates to a composition for plant disease control or growth promotion, containing Mariniflexile rhizosphaerae TRM1-10 or relative strains as an active ingredient. The composition comprises 16S rDNA of SEQ ID NO: 1, and can effectively control bacterial wilt.

Description

Technical Field [0001] The present invention relates to a composition for controlling plant diseases or promoting growth, which comprises a marine nipple leciliac sparra TRM1-10 or a near-

The present invention relates to a plant disease control or plant growth promoting composition comprising a newly isolated and identified Mariniflexile rhizosphaerae TRM1-10 (TRM1-10) as an active ingredient.

Bacterial wilt, one of the representative bacterial botanical bottles, is a disease caused by the green leaves and stems of plants. When leaf blight occurs, young leaves start to wilt in the early days. When the disease progresses rapidly according to the onset environment, the entire plant is wilted in a green state in 2-3 days, or if the plant is not worn out, it is brownish in the vascular system and a white streak is found.

The onset strain of foot blight, Ralstonia solanacearum ) is an aerobic bacterium with more than one flagella. Ralstonia Solanaceae rooms survive in soil for more than 4 years and enter plants through their roots. The plants that are infected with Ralstonia Solanacea Room are more than 50 industrial crops, including potatoes, tomatoes, bananas, peanuts, branches, peppers, tobacco, and many other industrial crops. Because it is transmitted to crops, it is spread quickly in large areas in the rainy season, and the damage of crop loss is great.

Various control methods have been developed for the pathogenic strain of foot rot, and the control method using chemically synthesized pesticide which directly removes the onset strain has been widely used. However, chemical synthetic pesticides contain a large amount of substances harmful to human body, and the harmful substances remain in soil and crops. Therefore, studies on effective treatment of foot blight without harming the human body are under way.

On the other hand, plants are symbiotic with various microorganisms present in the rhizosphere. Rootstock is a soil zone affected by the physiological action of plant roots, ranging from a few millimeters (mm) to a few centimeters (cm) from the root surface.

In rhizosphere soil, plant roots absorb carbon dioxide and various organic matter while absorbing moisture, inorganic nutrients, and oxygen.

Therefore, rhizosphere soil is distinguished from non - rhizosphere soil by organic matter content, microorganism and microbial density, soil pH, etc. Microorganisms in the rhizosphere coexist with each other in symbiosis with each other, and Ralstonia solanaceae, an onset strain of foot rot, is also a microorganism of these rhizomes.

However, even if the same kind of crops are infected with the same botanical pathogens, there are crops that have plant diseases and those that do not. In general, plant-specific genetic immune system affects plant disease outbreaks, but in addition to the inherent plant defense system, differences in rhizosphere environment that affect plant physiological activity may affect plant disease outbreaks.

In recent years, there has been an increasing study on whether a strain isolated from the rhizosphere of a plant tolerant crop can be used as a biological control. Biological control using microorganisms or plant extracts instead of chemical synthetic pesticides can selectively provide control of the target disease without affecting the human body. Therefore, it is possible to provide safe crops to consumers while reducing industrial losses. have.

The present inventors therefore intend to provide new strains and compositions that can be used as a preventive against foot wilt disease that has been isolated from crops resistant to foot wilt disease.

 International Journal of Microbiology, Vol. 2014, pp. 1-14 (May 19, 2014).

An object of the present invention is to isolate a new strain from the rhizosphere of a crop resistant to foot rot, and to identify a strain that has a novel control effect against foot rot disease.

It is an object of the present invention to provide a composition for controlling wilt blight comprising a strain or a culture thereof having an effect of preventing wilt disease as an effective ingredient.

It is an object of the present invention to provide a method for controlling foot wilt disease using the composition for preventing foot wilt disease.

According to an aspect of the present invention, there is provided a strain comprising the 16S rDNA of SEQ ID NO: 1 and having a controlling effect against a plant disease.

In one embodiment, the strain may be a Bacteroidetes door.

In one embodiment, the strain may be a Mariniflexile genus .

In one embodiment, the botanical bottle is selected from the group consisting of a plant wilt, a tomato bitter disease, a cabbage black disease, a red pepper bacterial spotty disease, a Chinese cabbage disease, a kiwi ulcer disease, a citrus ulcer disease, a peach bacterial peritonitis, a potato scab disease, A rot fungus, a potato broom bottle, a rice sheath blight, a tomato blight, a tomato wilt, a tomato root blight nematode, a rot fungus, a black rot fungus, a black rot fungus, an unloading bottle, One or more selected from the group consisting of cucumber root nematode, bean root nematode, red pepper pluck, lettuce sclerotin, ginseng spotted deciduous bottle, Chinese cabbage rootstock, vesicular disease, tobacco dressing disease, potato brown rot disease and ginseng root rot disease.

According to another aspect of the present invention, there is provided a marine leaf extract having a controlling effect against a plant disease, rhizosphaerae TRM1-10; KCTC 18646P).

According to another aspect of the present invention, there is provided a plant disease controlling composition comprising the strain or a culture thereof as an active ingredient.

In one embodiment, the botanical bottle is selected from the group consisting of a plant wilt, a tomato bitter disease, a cabbage black disease, a red pepper bacterial spotty disease, a Chinese cabbage disease, a kiwi ulcer disease, a citrus ulcer disease, a peach bacterial peritonitis, a potato scab disease, A rot fungus, a rot fungus, a potato broom disease, a rice sheath blight, a rice blast, a tomato plague, a tomato, a rot fungus, a black rot fungus, an unloading bottle, an incoherent bottle, a scarlet rot disease, a sclerotinia wilt, One or more selected from the group consisting of wilt disease, tomato root nodule, wheat red rust, barley powdery mildew, red pepper anthracnose, pepper pluck, lettuce sclerotinia, ginseng spotted deciduous bottle, cabbage root rot disease, wilt disease and cucumber root nematode.

According to another aspect of the present invention, there is provided a plant growth promoting composition comprising the strain or a culture thereof as an active ingredient.

According to another aspect of the present invention, there is provided a plant disease control method comprising the step of treating the plant disease control composition.

According to another aspect of the present invention, there is provided a method for producing a plant disease controlling composition comprising culturing the strain.

According to the present invention, the strain and composition for controlling foot wilt disease can effectively control foot wrinkling.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the details of the present invention or the composition of the invention described in the claims.

Fig. 1 shows the results of cultivating the mariniflexile TRM1-10 ( Mariniflexile) strain isolated from the rhizome of Hawaii 7996, a tomato variety tolerant to foot rot, rhizosphaerae TRM1-10; KCTC 18646P). (A) Colony grown at 30 ℃ for 4 days in marine agar medium. (B) Scanning electron micrograph of the strain TRM1-10 of marine nipple R. licorice sp. Grown in marine agar medium at 30 ° C for 4 days.
FIG. 2 shows the results of identification of the 16S rRNA gene sequence 1 of the TRM1-10 strain as a phylogenetic position.
FIG. 3 is a graph showing the effect of controlling TRM1-10 strains against foot wilt disease. Disease index of foot blight is 0, no disease symptoms; 1, 1-25% of leaves are wilted; 2, 26 ~ 50% of leaves are wilted; 3, 51 ~ 75% of leaves are wilted; 4, 76 ~ 100% of the leaves were wilted. The experiment for evaluating the prevention of foot blight disease using the strain TRM1-10 was repeated three times to display an error bar indicating the error range on the graph.
Fig. 4 is a graph showing the plant growth promoting effect of TRM1-10 strain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

When an element is referred to as " comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

Unless otherwise defined, can be performed by molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the art of recombinant DNA within the capabilities of the skilled artisan. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. The present invention is not limited to specific methods, protocols, and reagents, as it can be used in various ways depending on the context in which the person skilled in the art uses.

As used herein, the singular forms include plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are written from left to right, 5 'to 3', respectively, and the amino acid sequence is written from left to right, from amino to carboxyl.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

According to an aspect of the present invention, there is provided a strain comprising the 16S rDNA of SEQ ID NO: 1 and having a controlling effect against a plant disease.

The strain promotes resistance to plant diseases and inhibits the growth of microorganisms causing plant diseases, and thus can be usefully used as a microbial preparation for controlling plant diseases.

The plant diseases include plant diseases caused by a wide range of phytopathogenic bacteria, and include plant diseases such as foot rot, tomato ulcer disease, cabbage black disease, hot pepper bacterial spot disease, cabbage herpes disease, kiwi ulcer disease, citrus ulcer disease, peach bacterial pericarditis, Potato anthracnose, Scarab disease, Potato anthrax, Scarab disease, Perennial rot, Dry rot, Potato broom disease, Rice sheath blight, Tomato plague, Tomato blossom, Tomato rot, The group consisted of wilt disease, tomato root nematode, cucumber root nematode, soybean root nematode, red pepper pluck, lettuce sclerotin, ginseng spotted deciduous bottle, cabbage rootstock disease, wilt disease, tobacco dressing disease, potato brown rot disease and ginseng root rot disease More than one may be selected, but preferably it can be a foot blight.

The foot rot is caused by Ralstonia < RTI ID = 0.0 > solanacearum ) and may be, for example, but not limited to, foot rot, red pepper foot rot, foot rot, potato foot rot, paprika foot rot, or peanut foot rot.

The Mariniflexile subsp . Strains are resistant to Flavobacteriaceae ( Flavobacteriaceae ) belong to the family and can live in the marine environment, can live in the rhizosphere. The Mariniflexile genus was first isolated from the sea urchin of Troitsa Bay, Russia in 2006, and the Mariniflexile genus was identified as M. aquimaris , M. gromovii , M. ostreae , M. jejuense , M. soesokkakense , M. fucanivorans can be a total of six species.

According to another aspect of the present invention, there is provided a marine leaf extract having a controlling effect against a plant disease, rhizosphaerae TRM1-10; KCTC 18646P).

The above-mentioned strain of Mariniflex Rexiolis sp. TRM1-10 (hereinafter referred to as "TRM1-10") was deposited on November 28, 2017 in the Korean Collection for Type Cultures (KCTC) (Accession No .: KCTC 18646P) .

The Mariniflexile rhizosphaerae TRM1-10 (KCTC 18646P) is useful for the treatment of bacteria such as Bacteroidetes gland, Flavobacterias river, Flavobacteriaceae , Lek silica (Mariniflexile), a is a novel strain of rayijo spare below (rhizosphaerae) species.

The TRM1-10 strain is Gram-negative aerobic and is a bacterium, which does not form spores but performs sliding movement. The size is between 0.3 and 0.5 μm in diameter and between 3 and 4 μm in length.

The TRM1-10 strain forms convex yellow colonies within 3 days in an oceanic agar medium at 30 ° C, and the diameter of the colonies formed is 0.5-2 mm.

It does not occur in anaerobic agar agar medium for 10 days, growth temperature range is 4 ~ 40 ℃, and optimal growth temperature can be 30 ℃. The optimal growth pH of the strain is 7.0 to 8.0 and the optimal growth NaCl concentration may be 5% (w / v).

The strain TRM1-10 is negative for nitrate reduction, H2S production and oxidase activity, and positive for catalase activity. Flexirubin type pigment was not detected, and enzyme analysis through API ZYM and 20NE was as follows.

Enzyme activity positive: [alpha] -galactosidase, phosphatase, [beta] -galactosidase, [beta] -glucosidase, esterase (C4)), esterase lipase (C8), leucine arylamidase, N-acetyl-β-glucosaminidase, naphthol- AS-BI-phosphohydrolase, and valine arylamidase.

Enzyme activity negative: a-fucosidase,? -Glucosidase,? -Mannosidase,? -Chymotrypsin, arginine dihydro Arginine dihydrolase,? -Glucosidase,? -Glucuronidase, crystine arylamidase, lipase (C14), protease protease, trypsin, and urease.

Carbon sources such as D-cellobiose, D-mannose, D-raffinose, lactose, maltose, melibiose, succinate D-galactose, D-mannitol, D-ribose, L-arabinose, and the like were used. (L-arabinose), and L-rhamnose into carbon sources.

The antibiotic susceptibility and tolerance of Mariniflex Rizzo sp. TRM1-10 are as follows.

Antibiotic susceptibility: ampicillin, carbenicillin, lincomycin, novobiocin, rifampicin, and tetracycline. Antibiotic sensitivity: ampicillin, carbenicillin, lincomycin, novobiocin, rifampicin, and tetracycline.

Antibiotic resistance: cephalothin, gentamicin, kanamycin, neomycin, penicillin, polymyxin, streptomycin, and cycloheximide. ).

MK-6 (menaquinone-6) is the isoprenoid quinone of the marine niflex leciliac spraper TRM1-10 and iso-C _{15: 0} , iso-C _{17: 0} 3-OH and iso-C _{15: 0} G.

The polar lipids of the TRM1-10 strain are phosphatidylethanolamine, unidentified aminolipid and undetermined lipid.

The Mariniflexile genus can be identified by analyzing 16S rDNA of SEQ ID NO: 1.

The "16S rDNA (16S rDNA)" refers to DNA encoding 16S rRNA, and although there is almost no diversity in alleles, diversity among different species appears and is widely used for identification of the organism. The 16S rDNA is interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above.

The 16S rRNA gene analysis method has excellent advantages in clarifying phylogenetic relationships among bacteria or in identification of fungi. The inventors of the present invention have found that by analyzing 16S rDNA, a novel Mariniflex genus having excellent control activity against foot rot disease The strain was identified.

Specifically, it can be identified by amplifying the 16S rDNA of the strain through a primer.

The primer may be a short nucleic acid sequence including a free 3 'hydroxyl group and form a base pair with a template of the complementary nucleic acid, and a strand copy of the nucleic acid template Can be used as a starting point.

The primer can initiate DNA synthesis in the presence of a reagent for polymerization and a different four nucleoside triphosphate at the appropriate buffer solution and temperature and hybridize with the nucleic acid present in the target microorganism to produce a specific gene of the target microorganism Can be used for amplification.

In consideration of the efficiency of amplification, the primer may be single-stranded or deoxyribonucleotide.

The primers may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides, or non-natural nucleotides. In addition, the primer may also include ribonucleotides.

The primers include, but are not limited to, skeletal modified nucleotides such as peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, O-methyl RNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, And nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, 7-deazapurines having a C-7 substituent (the substituents being fluoro, bromo, chloro, iodo), such as methoxy, ethoxy, propyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl- -, methyl-, ethyl-, vinyl -, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.

The primer may be an extension primer that is annealed to the target nucleic acid to form a sequence complementary to the target nucleic acid by the template-dependent nucleic acid polymerase and extends to a position where the immobilization probe is annealed to anneal the probe Can occupy the site.

The extension primer may comprise a hybridization nucleotide sequence complementary to a specific position of the target nucleic acid.

The term " complementary " means that under certain annealing or hybridization conditions the primer or probe is sufficiently complementary to hybridize selectively to the target nucleic acid sequence, and is substantially complementary and perfectly complementary ≪ / RTI > The " substantially complementary sequence " may include not only a completely matched sequence but also sequences that partially overlap with the sequence to be compared, so long as the sequence can anneal to a specific sequence and serve as a primer.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. The " nucleic acid molecule " is meant to encompass both DNA (gDNA and cDNA) and RNA molecules. Nucleotide, the basic building block in nucleic acid molecules, (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

The specific sequence may be identified by various methods known in the art. For example, fluorescence nucleic acid hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, (Finkelstein et al., Genomics, 7: 167 (1990)), datablot analysis, denaturation gradient (see, for example, SSCA, Orita et al., PNAS, USA 86: 2776 (DGGE, Wartell et al., Nucl. Acids Res., 18: 2699 (1990)), a method using a protein recognizing nucleotidic mismatch (e.g., mutS protein of E. coli ) Ann. Rev. Genet., 25: 229-253 (1991)), and allele-specific PCR can be used.

Sequence changes can result in differences in base-linkage within the single-stranded molecule, resulting in the formation of bands with different mobility, and the SSCA can detect the band. The above DGGE analysis can detect a sequence showing different mobility from the wild type sequence using a modified gradient gel.

According to another aspect of the present invention, there is provided a plant disease control composition and a plant growth promoting composition comprising the strain or a culture solution thereof as an active ingredient.

The composition may be in the form of a wettable powder or a suspension concentrate. The composition for preventing wilt blight disease may be prepared in the form of a liquid, and an extender may be added to the powder to be used in the form of a powder, or may be formulated into granules, but is not limited thereto.

The composition may be uniformly diluted with water in sufficient amounts for plant disease control or promoting growth and then sprayed onto the arable land using a suitable spraying device such as a power sprayer.

When the wettable powder or the liquid wettable powder is diluted with water, the concentration of the wettable powder or the liquid wettable powder may be adjusted to about 10 ⁵ to 10 ¹⁰ CFU / ml, preferably about 10 ⁸ CFU / ml so that the effective ingredient may be in a biologically effective range , But is not limited thereto.

The composition may comprise an agriculturally acceptable carrier and may contain one or more additives such as fillers, solvents, excipients, surfactants, suspending agents, spreaders, adhesives, , Dispersants, wetting agents, drift reducing agents, auxiliaries, adjuvants or mixtures thereof.

The formulation of the composition is not particularly limited and may be formulated into concentrate, solution, spray, aerosol, immersion baths, dips, emulsions, suspension concentrates, gels, can do.

The composition may be used alone or in combination with other agricultural preparations, pesticides, insecticides, acaracides, fungicides, fungicides, herbicides, antibiotics, antimicrobials, nematicides, rodenticides Pheromones, attractants, plant growth regulators, plant hormones, insect growth regulators, chemosterilants, and the like), insect growth regulators, , Microbial pest control agents, repellents, viruses, phagostimulents, plant nutrients, plant fertilizers and biological control agents, or they may be used sequentially, but their use is particularly limited It is not.

The composition may be a fresh strain comprising the 16S rDNA of SEQ ID NO: 1 or a culture obtained by culturing the strain of Mariniflex Rexilai sp. Strain TRM1-10 in a marine agar medium and a marine liquid medium at 30 DEG C for 3 days.

The composition may be a suspension comprising the 16S rDNA of SEQ ID NO: 1 or a strain of Mariniflex Rexilis sp. Strain TRM1-10 diluted in purified water. The concentration of the suspension of the strain may be 2 x 10 ⁶ to 2 x 10 < ⁸ > CFU / g of soil.

According to another aspect of the present invention, there is provided a plant disease control method comprising the step of treating the plant disease control composition.

The composition for controlling plant disease can be treated by mixing with the plant soil of the crop to be treated, and the liquid composition can be injected onto the soil of the crop to be treated, or the soil mixed with the composition by separating the crop from the pollen But the method of use is not particularly limited.

The amount of the plant disease controlling composition varies depending on the target disease, the target crop, the application method, the occurrence tendency, the degree of damage, the environmental condition, the formulations to be used, and the like.

According to another aspect of the present invention, there is provided a method for producing a plant disease controlling composition comprising culturing the strain. The strains can be cultured by well-known methods in the art, and the cultured strains can be used for the preparation of compositions for controlling plant diseases.

Hereinafter, the present invention will be described in more detail by way of examples.

Experimental Method

Experimental Example  One : Foot dried  Separation of control strains from rhizosphere of resistant crops

Tomato varieties grown in the greenhouse of Dong - A University Test Package (N 35.239 °, E 128.978 °) were used to select strains for control of foot blight disease which can be used as a microbial agent to enhance resistance to foot rot.

Hawaii 7996 tomato varieties are tomato varieties resistant to foot rot, and were cultivated from October 14, 2011 to November 17, 2011. The cultivated soil conditions of tomato varieties are as follows.

pH 7.0; EC 39.50 dS / m; Salinity 2.53%; 2.62% organic matter; Total N 0.15 mg / kg; Total P 1,037.00 mg / kg; P ₂ O ₂ 545.26 mg / kg; Ca 1,688.00 kg / mg; Mg 216.50 mg / kg; K 106.90 mg / kg; Na 289.80 mg / kg; Total microbes 4.3 × 10 ⁷ CFU / g dry soil.

The strain TRM1-10 was isolated from the rhizosphere soil of the Hawaii 7996 tomato variety. Isolated TRM1-10 strains were cultured in marine agar medium and marine liquid medium at 30 ℃ and stored in 15% glycerol suspension at -80 ℃ for long term preservation.

Experimental Example  2 : TRM1 Morphological and biochemical analysis of -10 strains

Morphological and biochemical characteristics of TRM1-10 strains isolated from rhizosphere soil of tomato varieties of Hawaii 7996 were analyzed.

Gram staining kit (YD Diagnostics Inc., Korea) was used for Gram stain of TRM1-10 strain and morphology and motility of TRM1-10 strain were observed using optical microscope (Carl Zeiss, Axio Lab.A1).

In addition, a scanning electron microscope (FEI Quanta 250 FEG installed at Korea Research Institute of Bioscience) was used to observe the morphology and size of TRM1-10 strain (Fig. 1B).

Growth of TRM1-10 strains at temperatures ranging from 4 to 40 ° C was observed in marine agar medium for 7 days.

Growth of TRM1-10 strain in the range of 2 ~ 10% NaCl and 4.5 ~ 9 pH was observed for 7 days in a marine liquid medium at 30 ℃ temperature condition.

The anaerobic growth test of TRM1-10 strain was carried out in an anaerobic chamber for 10 days in an oceanic agar medium at 25 ° C.

The KOH test of TRM1-10 strain was judged by the presence or absence of a flexirubin pigment and the production of H ₂ S was tested with SIM medium and Kovacs reagent (Becton, Dickinson and Company, USA).

Enzyme activity of catalase and oxidase of TRM1-10 strain was tested using 3% H ₂ O ₂ and oxidase reagent (Becton, Dickinson and Company, USA).

API 20NE and API ZYM (bioMerieux, France) were used to investigate other enzyme activities and assimilation of TRM1-10 strains.

The casein hydrolysis test of TRM1-10 strain was carried out for 7 days in skim milk agar medium at 30 ° C containing 2% NaCl.

The starch, xanthine and hypoxanthine hydrolysis tests of strain TRM1-10 were carried out on marine agar medium containing 0.2% starch, 0.4% xanthine and 0.4% hyperxanthin.

The L-tyrosine hydrolysis test of TRM1-10 strain was carried out at 30 DEG C for 7 days in medium containing 0.5% peptone, 0.3% beef extract, 0.5% tyrosine and 2% NaCl.

95 assays of carbon source assimilation of TRM1-10 strain were carried out at 30 ° C for 3 days using Biolog GN2 microplates.

Antimicrobial susceptibility testing of TRM1-10 strains was performed on marine agar medium in an antibiotic susceptibility test disc (Oxoid, Thermo Fisher Scientific Inc., USA): ampicillin (10 μg / disc), carbenicillin 100 μg / disc), cephalothin (30 μg / disc), chloramphenicol (50 μg / disc), gentamicin (30 μg / disc), kanamycin (Nemocin, 30 μg / disc), novobiocin (5 μg / disc), penicillin (10 μg / disc), polymyxin (300 μg / disc), rifampicin (5 μg / disc), streptomycin (25 μg / disc), sulphamethoxazole (25 μg / disc), tetracycline Cycloheximide (100 μg / ml).

To compare the characteristics of chemical classification, Mariniflexile species were obtained from KCTC, and all strains were cultured in 30 ℃ oceanic agar medium and marine liquid medium.

To analyze fatty acids and polar lipids, strains TRM1-10 and M. soesokkakense RSSK-9 (KCTC 32427) were cultured in a 30 ° C agar medium for 3 days.

Fatty acids were extracted according to Miller's method and identified by gas chromatography (Agilent 6890 gas chromatograph, KCCM, South Korea).

50 mg of lyophilized TRM1-10 and M. soesokkakense RSSK-9 cells were prepared for polar lipid analysis. Polar lipids were extracted according to Miller's method and analyzed by 2-D thin-film chromatography (KCCM, South Korea).

100 mg of lyophilized TRM1-10 and M. soesokkakense RSSK-9 cells were prepared for isoprenoid quinine analysis. Isoprenoid quinine was extracted with chloroform-methanol (2: 1, v / v) and identified by high performance liquid chromatography (KCCM, South Korea).

The TRM1-10 strain was a gram-negative aerobic bacterium and formed yellow colony 0.5-2 mm in size on marine agar medium for 3 days (FIG. 1A).

The physiological and biochemical classification characteristics of TRM1-10 are shown in Table 1 below in comparison with M. soesokkakense RSSK-9.

Characteristic TRM1 -10 M. soesokkakense  RSSK-9 Growth temperature range (℃) 4-40 4-35 Nitrate reduction - + Oxidase activity - + Hydrolytic activity Casein - - Hypoxanthine - - Starch + + l-tyrosine - + Esculin + + Gelatin - - Tween 80 - - Use Carbon source Benzoate - - d-Cellobiose + - d-Galactose - + d-Mannose + + d-trehalose + - l-Arabinose - + 1-glutamate + - Maltose + + Succinate + - Sucrose + + Antibiotic  sensibility Ampicillin + + Carbenicillin + + Cephalothin - + Gentamicin - - Kanamycin - - Lincomycin + + Neomycin - - Novobiocin + + Polymyxin B - - Streptomycin - - Tetracycline + - DNA G + C ratio G + C (%) 34.56 34.6

Experimental Example  3: TRM1 -10 Molecular Biological Analysis of Strain

16S rRNA gene was amplified with the primer set of Table 2 for molecular biology identification. PCR conditions were annealing 55 ° C, 30 sec; extension at 72 ° C for 1.5 minutes and 30 cycles. The amplified 16S rRNA gene sequence was analyzed by Sanger sequencing and identified on EzTaxon web server.

Systematic analysis using the 16S rRNA gene was performed using ARB (Ludwing, W. et al., Nucleic Acids Res 32, 1363-1371, 2004) and MEGA5 (Tamura, K. et al., Mol Biol Evol 28, 2731-2739, 2011 ).

division primer  order 27F 5'-GTG CTG CAG AGA GTT TGA TCC TGG CTC AG-3 ' 1492R 5'-CAC GGA TCC TAC GGG TAC CTT GTT ACG ACT T-3 '

To analyze the G + C ratio of DNA, TRM1-10 DNA was extracted using Wizard Genomic DNA Purification Kit (Promega, USA) and analyzed using the PacBio RS II system of Illumina platforms (DNA Link, Inc., South Korea) The base sequence was analyzed using HiSeq 2000 sequencer.

The total length of 16S rRNA of TRM1-10 strain was 1,510 nt and the similarity of nucleotide sequence with M. soesokkakense RSSK-9 was the highest (96.94%).

In the 16S rRNA-based phylogenetic tree, TRM1-10 was included in the Mariniflexile genus and shared common ancestry with M. soesokkakense , M. fucanivorans and M. gromovii (Fig. 2).

As a result of comparing microbiology, genetics and biochemical characteristics with phylogeny and other Mariniflexile species, TRM1-10 strain is a new species of Mariniflexile genus. The present inventors tried to isolate TRM1-10 strain as Mariniflexile rhizosphaerae sp. nov.

Experimental Example  4 : TRM1 -10 Test for control of foot blight in strains

Tomato Moneymaker, a susceptible crop of foot blight, was used to evaluate the efficacy of TRM1-10 for control of foot wilt disease.

The surface-sterilized tomato seeds were germinated for one week in sterilized water and cultivated for 2 weeks in a pot containing soil (Punong Co., Ltd, Korea) used in a commercial horticultural facility sterilized twice at 40C for 12 minutes .

The crops used in the experiment were cultivated in a culture room controlled at 28 ℃ temperature condition for 14 hours under light condition and 10 hours under dark condition.

The strain TRM1-10 was cultured in a marine agar culture medium at 30 ° C for 4 days, and the suspension was prepared with sterilized water and treated in the pot-grown pot.

The suspension of the strain was treated with various dilution waters and mixed into the soil, adjusted to 2 × 10 ⁶ CFU / soil g, 2 × 10 ⁷ CFU / soil g and 2 × 10 ⁸ CFU / soil g per final soil. Tomato crops without strain suspension were used as a negative control, and all experiments were repeated 3 times with 10 tomatoes each.

All crops treated or not treated with TRM1-10 were cultured for 7 days under light condition for 14 hours and dark condition for 10 hours under temperature condition of 28 ℃, and R. solanacearum SL341, a pathogenic strain of foot rot, was cultured at 10 ⁷ CFU Were mixed in the soil for inoculation. Disease progression of foot blight was observed for 2 weeks after inoculation.

Tomato crops treated with TRM1-10 strain diluted at a high concentration (2 × 10 ⁸ CFU / soil g) showed a higher control effect against foot worms than tomato plants without TRM1-10 strain dilution 3).

Experimental Example  5: TRM1 Plant growth promotion test of -10 strains

The effects of TRM1-10 on the growth of tomato plants were investigated using natural farmland soil (Dae - dong University Donghae University Farm, Gimhae City, Gyeongsangnam - do).

In tomato varieties, Hawaii 7996 seeds were vigorously shaken in 70% ethanol with 0.1% Triton X-100 for 1 min, and then tomato seeds were vigorously shaken in 10% bleach (chlorox) supplemented with 0.1% Triton X-100 for 15 min Respectively.

The seeds were then harvested, thoroughly washed 5 times with sterile water, and dried to prepare sterile seeds. The soil was cultivated by mixing natural farmland and Calcined clay (washed in tap water and sterilized for 2 minutes at 121 ° C for 40 minutes) in a volume ratio of 1: 1.

The sterilized tomato seeds were grown in sterilized filter paper (Advantec; Toyo Roshi Co., Ltd., Tokyo, Japan) for 7 days at 32 ° C for 14 hours and 28 ° C for 10 hours.

Tomato seedlings germinated were divided into 60g portions per pot of farmland soil prepared on the 7th day after denture. The sterilized water or TRM1-10 suspension per pot was treated to control the number of bacteria per gram of soil to 10 ⁷ CFU.

After planting, the tomato plants were grown at 28 ° C for 14 hours and 28 ° C for 10 hours.

Forty days after the planting, tomato plants were firstly quantified and the fresh weight of the TRM1-10 strain and the untreated plants were measured and compared.

The repetition of each treatment was repeated with 5 plants, and the significance of each treatment was examined through T-test statistical processing.

The fresh weight of Hawaii 7996 was 2.418 g in TRM1-10 microbial treatment, which was 1.3 times higher than that of 1.86 g microbial control (p = 0.0039795) (Fig. 4).

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC18646P

Checked on: 20171128

<110> YONSEI UNIVERSITY <120> A COMPOSITION FOR CONTROLLING PLANT DISEASE AND PROMOTING PLANT          GROWTH COMPRISING MARINIFLEXILE RHIZOSPHAERAE TRM1-10 <130> 17PP10867 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1510 <212> DNA <213> Artificial Sequence <220> <223> TRM1-10_16S_rRNA <400> 1 tgaagagttt gatcctggct caggatgaac gctagcggca ggcctaacac atgcaagtcg 60 aggggtagag ggagcttgct cccttgagac cggcgcacgg gtgcgtaacg cgtatacaac 120 ctgcctctta ctgcgggata gcccagagaa atttggatta acaccgcata gtatagttac 180 ttggcatcaa gttattatta aaggttacgg taagagatgg gtatgcgttc tattagctag 240 atggagtggt aacggcacac catggcaacg atagataggg gccctgagag ggggatcccc 300 cacactggta ctgagacacg gaccagactc ctacgggagg cagcagtgag gaatattgga 360 caatgggggc aaccctgatc cagccatgcc gcgtgcagga agactgccct atgggttgta 420 aactgctttt gtacgggaag aaacactgcc acgtgtggca gcttgacggt accgtaagaa 480 taaggatcgg ctaactccgt gccagcagcc gcggtaatac ggaggatcca agcgttatcc 540 ggaatcattg ggtttaaagg gtccgtaggt ggatggttaa gtcagaggtg aaatcctgca 600 gctcaactgt agaattgcct ttgatactgg ctatcttgaa tcaatgtgaa gtggttagaa 660 tatgtagtgt agcggtgaaa tgcatagata ttacatagaa taccaattgc gaaggcagat 720 cactaacatt gcattgacac tgatggacga aagcgtgggg agcgaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatggatac tagctgttcg ggtttacctg agtggctaag 840 cgaaagtgat aagtatccca cctggggagt acgggcgcaa gcctgaaact caaaggaatt 900 gggggggcc cgcacaagcg gtggagcatg tggtttaatt cgatgatacg cgaggaacct 960 taccagggct taaatgtaag ttgcattagc tggagacagc tatttcttcg gaccacttac 1020 aaggtgctgc atggttgtcg tcagctcgtg ccgtgaggtg tcaggttaag tcctataacg 1080 agcgcaaccc ctgttgttag ttgccagcga gtcaagtcgg gaactctagc aagactgcca 1140 gtgcaaactg tgaggaaggt ggggatgacg tcaaatcatc acggccctta cgtcctgggc 1200 tacacacgtg ctacaatggt agggacagag agcagccact gggcgaccag gagcgaatct 1260 acaaacccta tcacagttcg gatcggagtc tgcaactcga ctccgtgaag ctggaatcgc 1320 tagtaatcgc atatcagcca tgatgcggtg aatacgttcc cgggccttgt acacaccgcc 1380 cgtcaagcca tggaagctgg gagtgcctga agtccgtcac cgcaaggagc ggcctagggt 1440 aaaatcggta actagggcta agtcgtaaca aggtagccgt accggaaggt gcggctggaa 1500 cacctccttt 1510

Claims (10)

delete delete delete delete Mariniflexile rhizosphaerae TRM1-10 (KCTC 18646P) strain having a control effect against foot rot disease. A composition for controlling wilt disease comprising the strain of claim 5 or a culture thereof as an active ingredient. delete A plant growth promoting composition comprising the strain of claim 5 or a culture thereof as an active ingredient. A method for controlling foot blight disease comprising the step of treating the composition of claim 6. A method for producing a composition for preventing wilt disease, comprising culturing the strain of claim 5.

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