KR100873962B1 - New Methylbacterium fuzisawaens CMB 110 strain and plant growth promotion method using same - Google Patents

Abstract

The present invention relates to a novel methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 110 strain and a method for promoting plant growth using the same. More specifically, the present invention is to fix aerial nitrogen and secrete plant growth promoting hormones. , Methylobacterium Methylobacterium Promotes Plant Growth by Inhibiting the Production of Plant Aging Hormones fujisawaense ) CBMB 110 strain and a method for promoting plant growth using the same. Plant growth promoting strain according to the present invention can be used to increase the productivity of the plant and to prepare microbial fertilizers using the strain.

Description

New Methylobacterium fujisawaense CBMB 110 and method of enhancing plant growth using Methylobacterium fujisawaense CBMB 110 strain

1 is a result showing the plant hormones increased in plants by the novel Methylobacterium fujisawaense CBMB 110 strain of the present invention.

Figure 2 is a result showing the amount of ethylene in the plant reduced by the novel Methylobacterium fujisawaense CBMB 110 strain of the present invention.

Figure 3 is a result showing the amount of 1-aminocyclopropane-1-carboxylic acid reduced by the novel Methylobacterium fujisawaense CBMB 110 strain of the present invention.

The present invention relates to a novel methylobacterium fujisawaense (110) CBMB 110 strain and a method for promoting plant growth using the same. More specifically, the present invention provides a method for fixing aerial nitrogen, secreting plant growth promoting hormones, Methylobacterium fujisawaense CBMB 110 strain that promotes plant growth by inhibiting plant aging hormone, and a method for promoting plant growth using the same.

Cultivation of crops using soil microorganisms is widely done in legumes. Microorganisms mainly used for the cultivation of such crops include Rhizobium ( Rhizobium ). When a legume is infected by this strain, it forms a root nodule and combines gaseous nitrogen (N₂) with hydrogen. Nitrogen fixiation is performed to convert the nitrogen to ammonia nitrogen (NH₃). Nitrogen fixation is of great agricultural importance. In the case of unfertilized soil, nitrogen deficiency is a natural result, but under these soil conditions, legumes with root nodules solve the problem of nitrogen deficiency themselves, so they can grow under soil conditions where plants cannot grow due to nitrogen deficiency. Stacey et al., Mol. Plant-Microbe Interact . 1991, 4: 332-340.

Among soil microorganisms, microorganisms that promote plant growth without having a symbiotic relationship with host plants include plant growth-promoting rhizobacteria (PGPR), which was discovered by Kloepper et al. In 1980. Examples of the above strains include Azospirrilum (Okon, 1985), Pseudomonas (Kloepper et al., 1980), Bacillus sp. [ Bacillus (Backman et al., 1994)], which fixate co-nitrogen. It is included as a microorganism. These plant growth-promoting microorganisms include a variety of methods such as fixation of aerial nitrogen (Boddy and Dobereiner, 1995), production of plant growth hormone, promotion of plant nutrient absorption (Hallmann et al., 1997), and suppression of plant disease (Pleban et al., 1995) It is known to help plant growth by eggplant function.

On the other hand, ethylene, a plant hormone, is known to be produced after plants convert methionine to 1-aminocyclopropane-1-carboxylic acid (ACC) via S-adenosylmethionine. It is known to be involved in the regulation of enemy pathways, affecting processes such as aging, flowering, fruiting, and fruit ripening. Accordingly, techniques have been developed for methods of inhibiting or reducing the synthesis of ethylene to delay plant aging and prolong life. In the related art, a novel gene encoding a protein having a function of regulating ethylene synthesis to promote root growth (Korean Patent No. 0443488), 1-aminocyclopropane-1-carboxylic acid which is a precursor of ethylene ( A composition comprising a synthase inhibitor of ACC), a method for prolonging the flower life of ornamental plants, accelerating and prolonging flowering in plants (Korean Patent No. 0271887) and N-acetyl amino for compounds that inhibit ethylene production. Oxyvinylglycine (AVG) and a composition comprising the same (Korean Patent No. 0562183).

However, the above contents are related to genes and compounds that inhibit ethylene synthesis, and there is no example applied to plant growth using microorganisms present in the root zone and the leaf zone of crops and having enzyme proteins that control ethylene of crops.

In agriculture, fertilizers are an important source of agricultural crops. However, when used too much, it may cause eutrophication of river water, water pollution of groundwater, imbalance of soil nutrients, and poor growth of crops, which may lead to a decrease in crop yield and a decrease in crop quality. However, the use of fertilizer to increase crop productivity is inevitable due to the reduction of agricultural land area due to the development and diversification of the industry. Accordingly, interest in developing eco-friendly functional agricultural materials using microorganisms is higher than conventional chemical fertilizers. have.

However, until now, there is no known strain which has a function of secreting a plant growth hormone, controlling ethylene synthesis, phytohormone fixing ability, and promoting plant growth.

Accordingly, the present inventors have isolated and identified a novel Methylobacterium fujisawaense CBMB 110 strain having the ability to fix aerial nitrogen from soil, and the novel strain of the present invention promotes plant growth. The present invention has been found to have a function of simultaneously secreting hormones, inhibiting hormones that promote aging of plants, and promoting plant growth.

Accordingly, an object of the present invention is to provide a novel Methylobacterium fujisawaense strain CBMB 110 which promotes nitrogen fixation, secretion of plant growth hormone, inhibition of plant aging hormone, and promoting plant growth. will be.

In addition, an object of the present invention is to provide a microbial preparation using the methylobacterium fujisawaense ( Methylobacterium fujisawaense ) CBMB 110 strain as an active ingredient.

In addition, another object of the present invention is to provide a method for promoting the growth of plants using the methylobacterium fujisawaense ( Methylobacterium fujisawaense ) CBMB 110 strain.

In order to achieve the object of the present invention as described above, the present invention is a novel methyllobacterium fuzisawaens ( Methylobacterium) that promotes nitrogen fixation, secretion of plant growth hormone, suppression of plant aging hormone and plant growth fujisawaense ) provides 110 CBMB strains.

In another aspect, the present invention provides a microbial agent comprising the methylobacterium fujisawaense CBMB 110 strain as an active ingredient.

In addition, the present invention provides a method for promoting the growth of plants using the Methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 110 strain.

Hereinafter, the present invention will be described in more detail.

Methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 110 strain of the present invention is characterized by promoting plant germination and growth through the immobilization of nitrogen and inhibition of plant growth hormone secretion and production of plant aging hormone have. Methylobacterium fujisawaense CBMB 110 strain of the present invention shows optimal growth at 28 ° C., pH 6.8, and has other physiological and biochemical properties as shown in Table 1 below.

TABLE 1

Characteristics CBMB110 Colony characters Reddish pink Cell shape non-spore-forming rods Size  0.56-0.72 × 2.213-2.367μm Gram reaction -Ve Motility + Cyst formation - PHB production + B chl- a activity + NaCl (0.5%) + NaCl (1.0%) + NaCl (2.0%) - NaCl (3.0%) - Temperature 20-30 ℃ + 4 and 40 ℃ - pH (optimum pH 6.8) + pH 5.0 to 10.0 + pH 4.0 - MR-VP test - Simmon citrate test + Indole production - H ₂ S production - Starch hydrolysis - Lipolytic activity W Casein hydrolysis - Gelatin hydrolysis - Cellulase activity - Oxidase + Catalase + Protease - mxaF gene PCR amplification + ACC deaminase + Pectinase - Cellulase - G + C content 69.2%

Methylobacterium fujisawaense CBMB 110 strain of the present invention has a nitrogen fixing ability to convert gaseous nitrogen to ammonium nitrogen in a hydrogen-bonded state, so that plants can grow even in a nitrogen-deficient condition. It was confirmed by measuring the acetylene reducing power known in the art (results not shown).

In addition, Methylobacterium fujisawaense CBMB 110 strain of the present invention is a hormone that promotes plant growth, IAA (called indole acetic acid or oxal), cytokine t-ZR (trans-zeatin Riboside) and iPA (isopentenyladenosine), which is shown to inhibit the synthesis of ethylene, which promotes the aging of plants (see FIG. 1). The inhibition of ethylene synthesis is inhibited by the synthesis of ethylene precursor ACC by 1-aminocyclopropane-1-carboxylic acid (ACC) deminase, the strain of the present invention has such ACC delamination activity It has been shown to inhibit the synthesis of ACC and ethylene in plants.

The present invention provides a microbial agent for promoting plant growth using the strain of the present invention. The microbial agent may include Methylobacterium fujisawaense CBMB 110 strain as an active ingredient. Microbial preparations according to the invention can be formulated for promoting plant growth in a conventional manner and can be prepared in the form of dry powder or liquid fertilizer. However, the formulation is not particularly limited. Preferably, it may be formulated as a plant growth promoting biofertilizer to replace the chemical fertilizer.

The present invention provides a method for promoting plant growth by using the new strain Methylobacterium fujisawaense CBMB 110. As a method of promoting the growth of the plant, a culture medium in which Methylobacterium fujisawaense CBMB 110 strain is cultured and a microbial agent using the strain are immersed in a seed or plant, or irrigated, that is, sprayed Can be done. In the case of the dipping method, the culture and the preparation may be poured into the soil around the plant or the seeds may be immersed in the culture and the preparation. In the case of spraying, it can be sprayed in a streamlined plant by techniques well known in the art.

Plants capable of promoting growth by the method of the present invention may include most of the plants, particularly effective in rice, tomatoes, canola, pepper, and the like.

Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not necessarily limited to these Examples.

<Example 1>

Isolation of Strains

<1-1> Isolation of Strains from Rice

In order to isolate strains in Oryza sativa L., Holland and Polacco (1992) were prepared using ammonium mineral salt minimal medium (AMS) after grinding the rice, with 0.5% methanol added as the only carbon source. Strains were isolated by the proposed method.

<1-2> Identification of Isolated Strains

The nucleotide sequence of 16S rDNA of the selected lactic acid bacteria was analyzed according to a conventional method known in the art. As a result, it was confirmed that the 16S rDNA nucleotide sequence of the lactic acid bacteria (SEQ ID NO: 1) is 99% identical to the 16S rDNA nucleotide sequence of methyllobacterium fuzisawaens.

 Therefore, the present inventors named the strain "Milobacterium fuzisawaens CBMB 110" and deposited it on January 6, 2006 with the Institute of Agricultural Biotechnology (Accession Number: KACC-91211P).

<Example 2>

Plant Growth Hormone Secretion and Cytokine Storage Capacity by New Bacteria CBMB 110

To determine how the strain of the present invention affects plant growth hormone in plants, 1 g of canola seedlings were added to a TBS solution (3.03 g of Trizma base, 5.84 g sodium chloride, 0.2 g magnesium chloride hydrate, 0.2 g sodium azide , pH 7.2). The homogenized solution was centrifuged at 3885 × g for 5 minutes and then centrifuged at 3885 × g for 3 minutes. Plant extract supernatants obtained by centrifugation were used to measure plant growth hormone IAA (oxine) and plant cytokines t-ZR (trans-zeatine riboside) and iPA (isopentenyladenosine). The measurement method was performed by the immunoassay method by the guide method using an ELISA test kit (AGDIA, USA). The amount of plant hormone (IAA) and cytokine in the plant extract obtained by centrifugation was confirmed by measuring the absorbance at 405 nm using an ELISA plate reader (Biorad Model 550, Japan) after color development. As a control, seedling control of canola treated with magnesium sulfate on seed instead of culture medium in which the new strain CBMB 110 was cultured, seedling of canola treated with AVG known as an ethylene inhibitor, and E. cloacae , a strain without ACC deminination activity UW4 / AcdS ^- the ^T and Mf of the seedlings treated with the culture broth was used as canola. As a result, as shown in Figure 1 concentrations of plant growth hormone IAA (oxine) and cytokines t-ZA and iPA involved in plant growth in plants treated with the strain of the present invention Methylbacterium CBMB 110 Was found to be higher than the control group.

<Example 3>

 1-Aminocyclopropane-1-carboxylic Acid (ACC) Deamiase Activity Measurement

In order to check whether the new strain of the present invention has the activity of ACC diamines, the new strain CBMB 110 of the present invention was cultured in DF minimal salt medium. At this time, 3 mM ACC was added to confirm that ACC was used as the nitrogen source. Subsequently, the amount of alpha-ketobutyrate produced by ACC cleavage by ACC diamine was measured using a spectrophotometer at an absorbance of 540 nm, and the concentration of protein was measured using a Lori method. As a control, Mf ^T , which is another strain of methyllobacterium fuzisawaens type, and the methyllobacterium fuzisawaens CBMB 130 isolated in Example 1 were used. The results are shown in Table 2 below.

TABLE 2

Strain ACC diazase activity (concentration of generated alpha-ketobutyrate) CBMB 110 24.74 ± 4.12 CBMB 130 - Mf ^T 5.23 ± 0.58

As a result, as shown in Table 1, the ACC delamination activity of the new strain CBMB 110 of the present invention was shown to be superior to the other control strains CBMB 130 and Mf ^T.

<Example 4>

Inhibitory Effect of Ethylene on Ethylene by CBMB 110

CBMB 110 culture medium (A _{600 nm} = 0.15), a new strain of the present invention, was absorbed on the surface of 30 canola seeds, a type of cabbage. The seeds were placed on sterile filter paper with water added in 120 ml vials and the amount of ethylene produced was measured for 7 days. The amount of ethylene produced in each vial was measured using gas chromatography (DS 6200, Donam Instruments, Korea). As a control, the seed strain was treated with magnesium sulfate instead of the culture cultured with the new strain CBMB 110, AVG, which is known as an ethylene inhibitor, and E. cloacae UW4 / AcdS ^- and Mf ^T , strains without ACC deminination activity. Treated with the culture medium was used. As a result, as shown in Fig. 2, the amount of ethylene in the seed treated with the culture culture of the new strain CBMB 110 of the present invention was much less than that of the culture solution of magnesium sulfate and E. cloacae UW4 / AcdS ⁻ . It was shown that this was almost the same amount of ethylene as treated with ethylene inhibitor (AVG).

Example 5

Inhibitory effect of ACC by the new strain CBMB 110

Tissues of plants frozen in nitrogen gas were used to confirm whether ACC, an ethylene precursor, was inhibited by the new strain CBMB 110 of the present invention. 1 g of frozen plant tissue was extracted with 80% methanol containing antioxidant (BHT) and incubated at room temperature for 45 minutes. Centrifuged at 2000 g at 20 ° C. for 15 minutes and the pellet was dissolved in 4 ml of methanol. The solution was evaporated in vacuo. The remaining residue was taken up with 2 ml of water and the amount of ACC contained in the solution was measured by the methods of Wachter et al. (1999) and Lizada et al. (1979). Controls were the same as in Example 4 above. As a result, as shown in FIG. 3, the amount of ACC in the plant tissue by the new strain CBMB 110 of the present invention was found to be a little higher than that of the ethylene inhibitor (AVG), but it was smaller than that of other controls. It was confirmed to exist.

<Example 6>

Promoting Plant Growth by New Bacteria CBMB 110

In order to measure the plant growth promoting effect of the methyllobacterium fuzisawaens CBMB 110 of the present invention was tested on the seeds of canola, a type of cabbage. First, the bacterial strain CBMB 110 was incubated in aerobic conditions until the rate log state in AMS medium, and then transferred to DF salt medium to which the nitrogen source ACC (3.0 mM) was added. The culture conditions were as described by Penrose and Glick (2003). The culture was incubated for 1 hour after treatment to the canola seeds. Thereafter, the cells were incubated for 5 days while repeating light and dark for 12 hours at a temperature of 20 ± 1 ° C., and the germination rate of germination on the surface of seeds was measured. In addition, the root length of the germinated canola seedlings was measured. As a control, the seeds of canola were treated with magnesium sulfate instead of the culture of CBMB 110, a new strain, treated with AVG, known as an ethylene inhibitor, and E. cloacae UW4 / AcdS, a strain without ACC deminination activity. ^- and Mf was used a ^T processes the cultured broth.

As a result, as shown in Table 3, the germination rate of the seed treated with the CBMB 110, a new strain of the present invention, showed a maximum of 91.67%, which was very excellent compared to other controls. The growth of roots was also much higher than that of other controls.

TABLE 3

Seed germination and root growth promoting effect of the strain of the present invention

process Germination rate (%) Root Length (cm) Control 86.67 ± 0.79 4.76 ± 0.31 Methyl Bacterium Fujisawa Enshi CBMB 110 91.67 ± 1.73 7.67 ± 0.27 AVG 78.33 ± 3.93 6.23 ± 0.16 UW4 / AcdS ^- 80.00 ± 1.89 4.98 ± 0.37 Mf ^T 88.33 ± 3.46 6.47 ± 0.32

As described above, the novel Methylobacterium fujisawaense CBMB 110 strain of the present invention has the ability to fix the nitrogen, promote the secretion of plant growth promoting hormones and promote the aging of plants It has the activity of inhibiting hormones. Therefore, the novel strain of the present invention has the effect of promoting the germination and growth of the plant can be used in the production of microbial fertilizers can be used to increase the growth and crop yield of crops replaced with chemical fertilizers. .

<110> SA, DONGMIN <120> Novel Methylobacterium fujisawaense CBMB 110 and method of          enhancing plant growth using the same <130> DPP060132KR <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1412 <212> DNA <213> Methylobacterium sp. <400> 1 gctcagagcg aacgctggcg gcaggcttaa cacatgcaag tcgagcggac ctttcggggt 60 cagcggcgga cgggtgagta acgcgtggga acgtgccttc cggttcggaa taaccctggg 120 aaactagggc taataccgga tacgccctta tggggaaagg tttactgccg gaagatcggc 180 ccgcgtctga ttagctagtt ggtggggtaa cggcctacca aggcgacgat cagtagctgg 240 tctgagagga tgatcagcca cactgggact gagacacggc ccagactcct acgggaggca 300 gcagtgggga atattggaca atgggcgcaa gcctgatcca gccatgccgc gtgagtgatg 360 aaggccttag ggttgtaaag ctcttttatc cgggacgata atgacggtac cggaggaata 420 agccccggct aacttcgtgc cagcagccgc ggtaatacga agggggctag cgttgctcgg 480 aatcactggg cgtaaagggc gcgtaggcgg cgttttaagt cgggggtgaa agcctgtggc 540 tcaaccacag aatggccttc gatactggga cgcttgagta tggtagaggt tggtggaact 600 gcgagtgtag aggtgaaatt cgtagatatt cgcaagaaca ccggtggcga aggcggccaa 660 ctggaccatt actgacgctg aggcgcgaaa gcgtggggag caaacaggat tagataccct 720 ggtagtccac gccgtaaacg atgaatgcca gctgttgggg tgcttgcacc gcagtagcgc 780 agctaacgct ttgagcattc cgcctgggag agtacggtcg caagattaaa actcaaagga 840 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgcagaa 900 ccttaccatc ctttgacatg gcgtgttacc cagagagatt tggggtccac ttcggtggcg 960 cgcacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020 gcaacgagcg caacccacgt ccttagttgc catcattcag ttgggcactc tagggagact 1080 gccggtgata agccgcgagg aaggtgtgga tgacgtcaag tcctcatggc ccttacggga 1140 tgggctacac acgtgctaca atggcggtga cagtgggacg cgaaggagcg atctggagca 1200 aatccccaaa agccgtctca gttcggattg cactctgcaa ctcgagtgca tgaaggcgga 1260 atcgctagta atcgtggatc agcatgccac ggtgaatacg ttcccgggcc ttgtacacac 1320 cgcccgtcac accatgggag ttggtcttac ccgacggcgc tgcgccaacc gcaaggaggc 1380 aggcgaccac ggtagggtca gcgactgggg tg 1412

Claims (5)

Methylobacterium fujisawaense CBMB 110 strain (KACC-91211P) having a nucleotide sequence of SEQ ID NO: 1 for promoting plant growth. [Claim 2] The novel Methylobacterium fujisawaense CBMB 110 strain (KACC-91211P) according to claim 1, wherein the plant is selected from the group consisting of rice, tomato, canola and red pepper. The microbial agent containing Methylobacterium fujisawaense CBMB 110 strain (KACC-91211P) of Claim 1 as an active ingredient. According to claim 3, wherein the microbial agent is a microbial agent, characterized in that the chemical fertilizer replacement biofertilizer. Plant growth promoting method comprising the step of immersing or irrigation of the strain of claim 1 in a plant or plant seeds.

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