KR101972067B1 - Pedobacter ginsengisoli T01R-27 promoting plant growth and inducing tolerance of plants to abiotic stress and uses thereof - Google Patents

Abstract

The present invention relates to a new strain of Pedobacter ginsengisoli T01R-27 which has an effect of promoting plant growth and inducing resistance to environmental stress in plants such as temperature or salts, and uses thereof. The treatment of the Pedobacter ginsengisoli T01R-27 strain to a plant promotes the growth of the plant and increases resistance to various environmental stress in the plant such as low temperature, high temperature, or soil salts so that the T01R-27 strain can be effectively used as an environmentally friendly microorganism preparation.

Description

[0001] The present invention relates to a pedobacter ginseng solae T01R-27 strain having a plant resistance-inducing effect on plant growth promotion effect and an environmental disorder, and a use thereof,

TECHNICAL FIELD The present invention relates to a new strain of Pedovar JEUNGSUN SOLAI T01R-27, which has an effect of promoting plant growth promotion and tolerance to plants against environmental disturbances such as temperature or salt, and its use.

Plants encounter a variety of environmental disturbances such as dry, high salt, cold weather, heavy metals, thermal shock, or ozone for a lifetime in a fixed environment. Such environmental disturbances, i.e., abiotic stresses, limit the growth and development of plants and reduce crop production such as plant material, seeds, fruit or other edible products.

In general, environmental disturbances affecting plants include climate-related disorders, air pollution, chemical disruptions and nutritional disorders. Examples of climate-related obstacles include droughts, floods, frosts, low temperatures, high temperatures, excessive light, or insufficient light. Air pollution can be in the form of carbon dioxide, carbon monoxide, sulfur dioxide, NOx, hydrocarbons, ozone, ultraviolet, or acid rain. Chemical disturbances can result from the application of insecticides, fungicides, herbicides, or heavy metals.

Cold weather is an impairment of plant species susceptible to cold temperatures, usually occurring in species of tropical or subtropical origin. Cold weather can cause discoloration or lesions on the leaves and can cause the plants to cool down when the roots cool down. At freezing temperatures, ice crystals can form in plants and can be fatal if ice crystals expand to protoplasts or remain for extended periods of time. In addition, under excessive high temperature stress, plant growth is generally inhibited, and in general, active plant tissues can hardly survive at temperatures above 45 ° C.

There is a continuing need for a method for promoting the growth of plants and for enhancing the tolerance of plants to various environmental disturbances. In order to prevent the growth of plants from being hindered by environmental disturbances such as low temperature, high temperature, or salt, there is a method of increasing nutrients absorbed by plants by improving soil nutrition. However, the development of a microbial agent that can promote the growth of plants in a environmentally friendly manner without using a chemical agent and promote the tolerance of plants against various environmental disturbances is insufficient.

Pedobacter ginsengisoli sp. nov., a DNase-producing bacterium isolated from a ginseng field in South Korea, Leonid N. Ten, International Journal of Systematic and Evolutionary Microbiology (2006), 56, 2565-2570

The present invention provides a composition for promoting plant growth and a method for promoting plant growth by isolating a new strain that can promote plant growth environmentally and using the same.

Further, a problem to be solved by the present invention is to isolate a new strain capable of inducing tolerance of plants against various environmental disturbances such as low temperature, high temperature, or salt, Which maintain or increase the survival and growth of the plant in the soil where the microorganism is integrated.

To achieve the above object, the present invention provides a Torpedo bakteo binary sengji solrayi (Pedobacter ginsengisoli) T01R-27 strain (KACC 92177P) in a plant tolerance inducing effect on the effectiveness and environmental disturbances promote plant growth.

The inventors of the present invention isolated and identified microorganisms from the rhizosphere of the tomatoes cultivated in Jeju Island. The activities of microorganisms (ACC deaminase activity, phosphate solubilization ability, auxin formation), which are known to help plant growth, And confirmed the resistance effect of plants against various environmental disturbances such as low temperature, high temperature, or salt soil, and named it Pedobacter ginsengisoli T01R-27 (KACC 92177P).

The strain Fedovar JEONGSUN SOLAI T01R-27 of the present invention was deposited with KACC (National Institute of Agricultural Science and Technology) on Apr. 14, 2017 and was assigned accession number KACC 92177P.

The present inventors analyzed the 16S rRNA gene sequence (FIG. 6, SEQ ID NO: 1) of the selected T01R-27 strain and found that the T01R-27 strain was a standard strain of Pedobacter ginseng isolate Gsoil 042 ^T ) and showed 98.2% homology, confirming that it was a Fedorberta jungsung solae species (Fig. 1).

The present invention provides a strain of Pedrobacter Jeungseong Solae T01R-27, a cell, a spore, a culture thereof, a dried product of such a culture or an extract of such a culture.

In the context of the present invention, the term " cell " is meant to include a cell wall fraction of a strain, a dead cell, or a dried cell. The culture medium in the culture broth can be removed and centrifuged or filtered to recover only the concentrated cells, and this step can be carried out according to the needs of the ordinary skilled artisan. The concentrated microbial cells can be preserved by freezing or freeze-drying according to a conventional method so as not to lose their activity.

As used herein, the term " extract of cultured product " means that the culture is extracted using various organic solvents so as to include various physiologically active substances, including, but not limited to, C _1-4 alcohol (for example, Methanol, ethanol, butanol, etc.), acetone, ethyl acetate and the like may be used alone, or two or more kinds of organic solvents may be sequentially used to carry out the extraction.

The strain Fedovar JungSeongSolai T01R-27 of the present invention can be cultured in a large amount by a conventional culture method of microorganisms of the genus Fedorobacter. As the culture medium, a medium composed of carbon source, nitrogen source, vitamins and minerals can be used. The medium for culturing may include both solid medium and liquid medium. For example, a solid medium such as nutrient agar (NA) or tryptic soy agar (TSA) can be used. As the liquid medium, nutrient broth (NB), King's medium B broth (KB), peptone sucrose broth (PSB), or tryptic soy broth (TSB). As a preferable medium, NB medium can be used. The culture can be carried out under ordinary culture conditions.

In addition, the present invention provides a plant growth promoting composition comprising a strain of the genus Pseudomonas strain T01R-27 as an active ingredient. In the present invention, the term " plant growth (or growth) " refers to the differentiation, flowering, development, size, weight, or quantity enhancement of plant germination, organs (roots, stems, leaves, .

The plant growth promoting composition of the present invention can be applied to various plants and can exhibit excellent growth promoting effect on applied plants. The plant may include a terminal leaf or a twin leaf plant, and may be any one or more plants selected from the group consisting of tobacco, cucumber, pepper, tomato, Chinese cabbage, wheat and barley.

In addition, the present invention provides a composition for inducing tolerance of plants against environmental disturbance, which comprises a strain of Fedornia jungensisolai T01R-27 as an active ingredient. In the present invention, the term " environmental tolerance (stress) tolerance " means that the survival or growth of a plant is maintained with respect to soil salt accumulation, low temperature, or high temperature stress that may occur during plant growth.

Preferably, the environmental disorder in which the strain of the present invention is resistant to inducing resistance to the strain < RTI ID = 0.0 > T. < / RTI > In Example 3 of the present invention, the growth of plants was measured under various salt conditions of 70, -500, and -1000 kPa, and the plants treated with the strain T01R-27 significantly promoted the growth of plants as compared with the untreated control, The strain T01R-27 was found to be superior to the salt tolerance inducing effect against salt stress.

In addition, preferably, the environmental disorder in which the strain of the present invention is resistant to inducing resistance to a strain of the genus Typhimurium T01R-27 of the present invention can be low-temperature or high-temperature stress. In Example 4 of the present invention, the growth of plants was measured at low temperature or high temperature. Plants treated with T01R-27 strain were significantly accelerated to grow plants even at low or high temperature compared with the untreated control, and T01R-27 strain It was confirmed that the resistance inducing effect of the plant against low temperature or high temperature stress was excellent.

In addition, cells, spores, cultures thereof, dried products of such cultures, or extracts of such cultures of the strain Fedovar Jens JS Solaris T01R-27 may be replaced or added to strains of the above-mentioned Fedorjchner Jensensolai T01R-27 strain have. Whether or not one or more of the live bacteria, strains, microorganisms, cells, spores, cultures thereof, the dried products of such cultures, or the extracts of such cultures of the strain Fedovar JEJUNG JOSE SOLAI T01R-27 is selectively treated may be determined depending on the seedling environment, And the like can be appropriately selected by a person skilled in the art.

The composition of the present invention may contain one or more selected from among the strains of Fedorpacin Jenseng Solae T01R-27, cells, spores, cultures thereof, dried products of such cultures, or extracts of such cultures.

The composition for inducing tolerance to plants against environmental disturbance of the present invention can be utilized as a plant tolerance inducing and growth promoting agent against various environmental disturbances. Such formulations may include, for example, fertilizers, seed coatings, soil conditioners, and the like. The preparation according to the present invention may be prepared in various forms and may be prepared, for example, in the form of a wettable powder, a granule, a capsule, or an emulsion for stable formulation.

The present invention also provides a plant growth promoting method comprising the step of spraying or applying the composition for promoting plant growth. The present invention also provides a method for inducing tolerance of a plant to an environmental disorder, which comprises spraying or applying a composition for inducing tolerance to plants against the environmental disorder.

The strain Fedovar Jensengisolai T01R-27 of the present invention can be used for the treatment of, for example, whole plant, root treatment, plant seed treatment, plant tissue treatment in breeding period, plant disease prevention before planting or drinking water, And a method of treating the soil in the vicinity of the plant or the plant by various methods known in the art without limitation thereto. In addition, the treatment of the above plants or plants around the soil can be carried out by various methods known in the art such as spraying, coating, cross-linking, infiltration, spraying, injection, coating, dusting or soaking, T01R-27 can be suitably selected by a person skilled in the art in consideration of the condition of the strain, the seedling setting environment, the season, the kind of the target plant, or the economy.

In addition, the present invention provides a method for producing a plant growth promoting composition comprising culturing a strain of Fedorpacin JungSeongSalai T01R-27. The culture method and the method for producing the composition of the Geneaceae Solae T01R-27 strain can be any method known in the art, and the method is not particularly limited.

Treatment of plants with the strain Fedovacten Jeungseong Solei T01R-27 of the present invention promotes the growth of plants and increases tolerance of plants against various environmental disturbances such as low temperature, high temperature or soil salts. Therefore, T01R-27 strain It can be effectively utilized as an environmentally friendly microorganism preparation.

FIG. 1 shows the result of analysis of 16S rRNA nucleotide sequences of T01R-27 strain and phylogenetic analysis of other highly pathogenic strains of Fedorberta.
FIG. 2 is a result of comparing the growth of tomato plants with that of untreated plants after the soil salt stress was treated on tomato plants treated with strain T01R-27 in Example 3. FIG.
FIG. 3 is a graph comparing live weights of tomato plants and untreated plants treated with strain T01R-27 in Example 3. FIG.
FIG. 4 shows the results of comparing the growth of plants after treatment with various stresses of T01R-27 treated plants in Example 4 against untreated plants.
FIG. 5 is a graph comparing the fresh weight of plants treated with the strain T01R-27 and the untreated plants in Example 4. FIG.
6 is a 16S rRNA gene base sequence of strain T01R-27.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1: Classification of T01R-27 strain

The phylogenetic location and identification of T01R-27 strain was performed using 16S rRNA sequencing. Genomic DNA extraction kit (Inclone, Korea) was used for genomic DNA extraction. The 16S rRNA gene was amplified using general purpose primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). Amplification products were analyzed with the Genotech (Daejeon, Korea) and sequenced with a 3100 Genetic Analyzer (Applied Biosystems, USA). That is, the 16S rRNA gene was sequenced using three primers (518F; CCAGCAGCCGCGGTAATACG, 800R; TACCAGGGTATCTAATCC, 984F; ACGCGARGAACCTTAC). The nucleotide sequences obtained were sequenced and linked using Seqman software (DNASTAR, USA). The 16S rRNA gene sequence of the T01R-27 strain in the present invention is SEQ ID NO: 1, which is shown in FIG. The sequence similarity of the 16S rRNA gene was analyzed by EzTaxon (http://www.ezbiocloud.net/eztaxon). For the preparation of the genealogy, the nucleotide sequence was aligned with the Clustal W program of the MEGA 6.0 program and the nucleotide sequence length was adjusted. Neighbor-joining algorithm was used to construct the system diagram, and the stability of the system was confirmed by performing bootstrapping 1000 times.

As a result of the phylogenetic analysis, the strain T01R-27 was identified as a pedobacterium jeansensisolai (Fig. 1) because the strain T01R-27 has 98.2% similarity to the gene of the 16S rRNA sequence, which is the same as that of the Fedorobacter jeansilsoni Gsoil 042 ^T (standard strain).

Example 2: Evaluation of the physiochemical characteristics of the strain T01R-27 and the activity of enzymes involved in plant growth promotion

Biochemical biochemical tests were conducted according to the manufacturer's instructions using API 20E, API 32GN (bioMerieu, France). The activity of the plant-growth-promoting enzymes formed by the strain J01J-27 from Fedovactor jinjungseolai was evaluated. To verify this, strain T01R-27 was cultivated in R2A broth at 28 ° C, and the growth of microorganisms was measured at an absorbance of 600 nm after 48 hours.

In order to evaluate the auxin production ability of T01R-27 strain, T01R-27 strain was cultured for 24 hours, and only bacterium was harvested through centrifugation. To the medium which was mixed with 1/2 TSB and tryptophan 100 ug / ml for 24 hours Lt; / RTI > After centrifugation at 4000 rpm for 10 minutes, 40 μl of supernatant was mixed with 80 μl of Salkowski solution (35% perchloric acid, 1 ml of 0.5 M FeCl ₃ ). Then, 2 μl of 10 mM phosphoric acid (1/40 dilution) was added and cultured at 10 ° C for 24 hours, and the auxin production was measured at 530 nm for absorbance.

(10 g of glucose, 5 g of Ca ₃ (PO ₄ ) ₂ , 0.5 g of (NH ₄ ) ₂ SO ₄ , 0.2 g of NaCl, and 10 g of NaCl were added to the PVK medium 0.1 g of MgSO ₄ .7H ₂ O, 0.2 g of KCl, 0.5 g of yeast extract, 0.002 g of MnSO ₄ .H ₂ O, 0.002 g of FeSO ₄ .7H ₂ O, 15.0 g of Agar, 1000 ml of distilled water, bromo-phenol- g, pH 7.0) for 2 weeks.

1-aminocyclopropane-1-carboxylic acid (ACC) To evaluate the deaminase activity, T01R-27 strain was cultured as described above, ACC (30 μmol / plate) was plated on DF minimal medium and a drop of bacterial suspension was dropped . Table 1 below shows the results of evaluating microorganism activity (ACC deaminase activity, phosphate solubilization ability, auxin formation) known to help survival or growth of plants in various environmental disturbance conditions against T01R-27 strain (+ activity , There is no activity). The morphological and biochemical characteristics of T01R-27 strain were also shown.

division characteristic Characteristics related to crop growth promotion Auxin production, phosphate solubilization ability - Volatile substance, ACC deaminase + Morphological characteristics Cell size (diameter ~ width) 0.43 ± 0.38 to 0.43 ± 0.05 Cell morphology Coccobacilus Colony size (3 days culture) 1.15 ± 0.12 Colony color White Growth environment Growth temperature range 4-28 ° C NaCl tolerance 0-2% Carbon availability D-glucosamine, N- acetyl-D-glucosamine, D-maltose, D-melibiose, D-mannose, D-saccharose, + 3-hydroxybutyric acid, 4-hydroxybenzoic acid, adipic acid, capric acid, D-mannitol, D-ribose, D-sorbitol, glycogen, inositol, itaconic acid, L-alanine, L-arabinose , L-fucose, L-histidine, L-proline, L-rhamnose, L-serine, malic acid, phenylacetic acid, potassium 2-ketogluconate, potassium 5-ketogluconate, potassium gluconate, propionic acid, sodium acetate, sodium malonate, acid, trisodium citrate, valeric acid - Other Asculin hydrolysis, β-galactosidase (PNPG) + Arginine dihydrolase, gelatin hydrolysis, glucose fermentation, indole production, nitrate reduction, urease -

As a result, it was confirmed that T01R-27 was able to hydrolyze acsulin and produce β-galactosidase by using seven carbon sources such as D-glucose. Growth conditions were able to grow at a temperature of 4-28 ℃ and NaCl up to 2%. ACC deaminase activity and volatiles production ability related to plant growth were also detected.

Example 3: Inducible effect of plant resistance against salt stress of strain T01R-27

After treatment with T01R-27 strains and non-treated strains in the greenhouse conditions, tomatoes were treated with various concentrations of salts and the growth of tomatoes was compared (FIG. 2). For this purpose, tomato seeds (variety: Juiken) were planted in plastic pots with a diameter of 9 cm and cultivated in a greenhouse for 7 to 8 days. When cotyledon emerged, 20 ml of bacterial suspension (OD600 = 0.5) was cultivated for 7 ~ 8 days and treated with salt. Composite salts were prepared from solutions with the treatments of 70, -500, and -1000 kPa, and treated with 20 ml of 3 times at 2 - day intervals. The plants were weighed after 2 weeks of planting.

As a result, in general, the untreated plants are inhibited from growth under the various salt conditions of 70, -500, and -1000 kPa, especially in the high salt condition. In contrast, when T01R-27 strain is treated, tolerance to salt conditions And the fresh weight of the plant was statistically significantly increased. In the case of -70 kPa, the relative increase was 12.0%, -51 kPa, 65.1% and -1000 kPa, respectively, compared with the untreated control (FIG.

Example 4: Inducible effect of plant resistance against temperature stress of strain T01R-27

Plants were grown and treated with T01R-27 strain under greenhouse conditions similar to the salt stress described above. After that, the plants were stressed for 5 days in a plant growth chamber adjusted to 10, 25, 40 ° C, and then grown for 2 weeks in a greenhouse condition to evaluate the growth of plants (FIG. 4).

As a result, when T01R-27 strain was treated, fresh weight was increased at low temperature (15.3%) at 25 ° C, 26.9% at 25 ° C and 42.5% at high temperature (40 ° C) 5).

Example  5: Statistical analysis

R agricolae package was used for analysis of variance and multiple gestation between the treatments. Multiple assays used a Least Significance Difference (LSD) test at P < 0.05, and all experiments were performed two or more times.

National Institute of Agricultural Science and Technology Microbial Bank KACC92177P 20170414

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Pedobacter ginsengisoli T01R-27 promoting plant growth and          inducing tolerance of plants to abiotic stresses and uses thereof <130> 2017-0518-10-A, P17-228 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1463 <212> RNA <213> Artificial Sequence <220> <223> Pedobacter ginsengisoli T01R-27 16S rRNA <400> 1 atggctcagg atgaacgcta gcggcaggcc taatacatgc aagtcgaacg ataccatctg 60 gcttgccaga tgggaaagtg gcgcacgggt gcgtaacgcg tatgcaacct accttaatca 120 gggggatagc ccgaagaaat tcggattaac accgcataaa aacacaggat agcattatct 180 aatgttcaaa tatttatagg attaagatgg gcatgcgtgt cattagctag ttggcggggt 240 aacggcccac caaggcgacg atgactaggg gatctgagag gatgaccccc cacactggta 300 ctgagacacg gaccagactc ctacgggagg cagcagtaag gaatattggt caatggaggg 360 aactctgaac cagccatgcc gcgtgcagga agacggccct ctgggttgta aactgctttt 420 attcgggaat aaaccacatt acgtgtaatg tgctgaatgt accgaaggaa taaggatcgg 480 ctaactccgt gccagcagcc gcggtaatac ggaggatcca agcgttatcc ggatttattg 540 ggtttaaagg gtgcgtaggc ggcttattaa gtcaggggtg aaagacggtg gctcaaccat 600 cgcagtgccc ttgatactga tgagcttgaa tggactagag gtaggcggaa tgtgacaagt 660 agcggtgaaa tgcatagata tgtcacagaa caccgattgc gaaggcagct tactatggtt 720 ttattgacgc tgaggcacga aagcgtgggg atcaaacagg attagatacc ctggtagtcc 780 acgccctaaa cgatgaatac tcgctgttag cgatatacag ttagcggcta agcgaaagcg 840 ttaagtattc cacctgggga gtacgcccgc aagggtgaaa ctcaaaggaa ttgacggggg 900 cccgcacaag cggaggagca tgtggtttaa ttcgatgata cgcgaggaac cttacccggg 960 cttgaaagtt agtgaatgat ctagagatag gtcagtgagc aatcacacga aactaggtgc 1020 tgcatggctg tcgtcagctc gtgccgtgag gtgttgggtt aagtcccgca acgagcgcaa 1080 cccctatgtt tagttgccag cacgttaagg tggggactct aaacagactg cctgtgcaaa 1140 cagagaggaa ggaggggacg acgtcaagtc atcatggccc ttacgtccgg ggctacacac 1200 gtgctacaat ggatggtaca gagggcagca agctggcaac agcaagcgaa tctcaaaaag 1260 ccattcacag ttcggataga ggtctgcaac tcgacctctt gaagttggat tcgctagtaa 1320 tcgcgtatca gcaatgacgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcaa 1380 gccatggaag ttgggggtac ctaaagtatg taaccgcaag gagcgtccta gggtaaaacc 1440 gataactggg gctaagtcgt aac 1463

Claims (9)

It promotes the tomato growth promotion effect and the tomato tolerance effect to the environmental disorder,
The environmental disturbance may be low or high temperature stress, or salt stress
Torpedo bakteo binary sengji solrayi (Pedobacter ginsengisoli) T01R-27 strain (KACC 92177P). (KACC 92177P), a culture solution thereof or a culture medium extract thereof as an active ingredient. A tomato growth promotion method comprising the step of spraying or applying the tomato growth promoting composition of claim 2 to a plant. delete A method for producing tomato composition for promoting growth, comprising the step of culturing a strain of Fedorpacin Jenseng Solae T01R-27 (KACC 92177P). (KACC 92177P), a culture solution thereof or a culture medium extract thereof as an active ingredient,
A composition for inducing tolerance of tomato against environmental disturbances such as low temperature or high temperature stress, or salt stress. delete delete A composition for inducing tolerance of tomato against environmental disturbance according to claim 6,
A method for inducing tolerance of tomatoes against environmental disturbances such as low or high temperature stress, or salt stress.

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