KR100873961B1 - Novel 20 strains of Methylbacterium fuzisawaens CMBM and methods for promoting plant growth using the same - Google Patents

Abstract

The present invention relates to a novel Methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 20 strain and a method for promoting plant growth using the same. More specifically, the present invention is to fix the nitrogen and secrete plant growth promoting hormone , Methylobacterium Methylobacterium Promotes Plant Growth by Inhibiting the Production of Plant Aging Hormones fujisawaense ) CBMB 20 strain and a method for promoting plant growth using the same. Plant growth promoting strain according to the present invention increases the productivity of the plant and can be usefully used for the production of microbial fertilizers using the strain.

Description

Novel Methylobacterium fujisawaense CBMB 20 and method of enhancing plant growth using Methylobacterium fujisawaense CBMB 20

1 is a result showing the plant hormones increased in plants by the novel Methylobacterium fujisawaense CBMB 20 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 20 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 20 strain of the present invention.

The present invention relates to a novel Methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 20 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 ( Cethylobacterium fujisawaense ) CBMB 20 strains that inhibit plant aging hormones to promote plant growth, and a method of promoting plant growth using the same.

Cultivation of crops using soil microorganisms is widely done in legumes. A microorganism mainly used for cultivating such crops is Rhizobium . When a legume is infected by this strain, root nodule is formed and gaseous nitrogen (N₂) is combined with hydrogen. Nitrogen fixiation is carried out to convert 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. The above strains are representative of the azosperilum fungi [ Azospirrilum (Okon, 1985)], Pseudomonas (Kloepper et al., 1980), Bacillus genus [ Bacillus (Backman et al., 1994), etc.). 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 ( N-acetyl amino for compounds that inhibit the ethylene production and methods of prolonging the flower life of decorative plants, accelerating and prolonging flowering in plants with compositions comprising inhibitors of synthase activity of ACC) Ethoxyvinylglycine (AVG) and compositions comprising the same (Korean Patent No. 0562183).

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

Therefore, the present inventors have isolated and identified a novel Methylobacterium fujisawaense CBMB 20 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 CBMB 20 strain that promotes nitrogen fixation, secretion of plant growth hormone, suppression of plant aging hormone, and promote plant growth. will be.

In addition, an object of the present invention is to provide a microbial preparation comprising the Methylobacterium fujisawaense CBMB 20 strain as an active ingredient.

In addition, another object of the present invention is to provide a method for promoting plant growth using the Methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 20 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 20 strains of CBMB.

In another aspect, the present invention provides a microbial agent comprising the Methylobacterium fujisawaense CBMB 20 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 20 strain.

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

Methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 20 strain of the present invention is characterized by promoting plant germination and growth through the immobilization of nitrogen and the inhibition of plant growth hormone secretion and production of plant aging hormone have. Methylobacterium fujisawaense ( Cethylobacterium fujisawaense ) CBMB 20 strain of the present invention shows an optimal growth at 28 ℃, pH 6.8 and other physiological and biochemical properties as shown in Table 1 below.

TABLE 1

Characteristics CBMB20 Colony characters Reddish pink Cell shape non-spore-forming rods Size 0.60-0.80 × 2.10-2.75 μm 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 70.6%

Methylobacterium fujisawaense CBMB 20 strain of the present invention has a nitrogen fixing ability to convert gaseous nitrogen into 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.

In addition, Methylobacterium fujisawaense CBMB 20 strain of the present invention is IAA (called indole acetic acid or oxal), a hormone that promotes plant growth, and 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 (see FIGS. 2 and 3).

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 20 strain as an active ingredient. Microbial preparations according to the present 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 the growth of plants using the new strain Methylobacterium fujisawaense CBMB 20. As a method of promoting the growth of the plant, a culture medium cultured with Methylobacterium fujisawaense CBMB 20 strain and a microbial agent using the strain are immersed in a seed or plant, or irrigated, that is, sprayed by 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 all plants, and are particularly effective for 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 base 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 20" and deposited it on 6 January 2006 with the Institute of Agricultural Biotechnology (Accession Number: KACC-91209P).

<Example 2>

Plant Growth Hormone Secretion and Cytokine Storage Capacity by New Bacteria CBMB 20

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 carried out by an enzyme immunoassay method by an instructional 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. A control group, is novel strain CBMB 20 The seedlings of being treated with magnesium sulfate to the culture broth instead of seed canola (control), it will be treated with AVG known ethylene inhibiting agent canola seedlings of, ACC dia laminate Ease in no active strain E It was used for ^T and Mf of the seedlings treated with the culture solution cultured canola ^- cloacae UW4 / AcdS. As a result, as shown in FIG. 1, the plant growth hormone IAA (indole acetic acid) and the cytokines involved in plant growth, t-ZA and iPA, in plants treated with the strain of the present invention Methylbacterium CBMB 20 as shown in FIG. It was confirmed that the concentration was 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 1-aminocyclopropane-1-carboxylic acid (ACC) diamine, the new strain CBMB 20 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 20 94.48 ± 6.83 CBMB 130 - Mf ^T 5.23 ± 0.58

As a result, as shown in Table 2, the ACC delamination activity of the new strain CBMB 20 of the present invention was found to be superior to the other control strains CBMB 130 and Mf ^T.

<Example 4>

Inhibitory Effect of Ethylene on Ethylene by CBMB 20

The culture medium (A _{600 nm} = 0.15) cultured with CBMB 20, 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 20, treated with AVG, known as an ethylene inhibitor, and E. cloacae UW4 / AcdS ^- and Mf ^T , strains without ACC deamiase activity. Treated with the culture medium was used. As a result, as shown in Figure 2, the amount of magnesium is ethylene in a novel strain of CBMB 20 of the present invention treated with the seed culture broth sulfate and E. cloacae UW4 / AcdS ^- ethylene in the process of the seed culture medium Much less than the amount appeared, which was almost the same as the treatment with ethylene inhibitor (AVG).

Example 5

Inhibitory effect of ACC by new strain CBMB 20

Tissues of plants frozen in nitrogen gas were used to confirm that the ethylene precursor ACC was inhibited by the new strain CBMB 20 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 Figure 3, the amount of ACC in the plant tissue by the new strain CBMB 20 of the present invention was found to be a little higher than the treatment with ethylene inhibitor (AVG), but less than other controls It was confirmed to exist.

<Example 6>

Promoting Plant Growth by New Bacteria CBMB 20

In order to measure the plant growth promoting effect of the methyllobacterium fuzisawaens CBMB 20 of the present invention was tested on the seeds of canola, a type of cabbage. First, the new strain CBMB 20 was incubated in aerobic conditions until the late log state in AMS medium, and then transferred to the 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. 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 20, 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 CBMB 20, the new strain of the present invention, was up to 96.67%, 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 ± .31 Methyl Bacterium Fujisawa Enshi CBMB 20 96.67 ± 2.67 8.50 ± 0.47 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 20 strain of the present invention has the ability to fix aerial nitrogen, promote the secretion of plant growth hormone, 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 plants can be used for the production of microbial fertilizers can be used to increase the yield of crops and the growth of crops replaced with chemical fertilizers.

<110> SA, DONGMIN <120> Novel Methylobacterium fujisawaense CBMB 20 and method of          enhancing plant growth using the same <130> DPP060112KR <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1416 <212> DNA <213> Methylobacterium sp. <400> 1 tcagagcgaa cgctggcggc aggcttaaca catgcaagtc gagcggacct ttcggggtca 60 gcggcggacg ggtgagtaac gcgtgggaac gtgccttccg gttcggaata accctgggaa 120 actagggcta ataccggata cgcccttatg gggaaaggtt tactgccgga agatcggccc 180 gcgtctgatt agctagttgg tggggtaacg gcctaccaag gcgacgatca gtagctggtc 240 tgagaggatg atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc 300 agtggggaat attggacaat gggcgcaagc ctgatccagc catgccgcgt gagtgatgaa 360 ggccttaggg ttgtaaagct cttttatccg ggacgataat gacggtaccg gaggaataag 420 ccccggctaa cttcgtgcca gcagccgcgg taatacgaag ggggctagcg ttgctcggaa 480 tcactgggcg taaagggcgc gtaggcggcg ttttaagtcg ggggtgaaag cctgtggctc 540 aaccacagaa tggccttcga tactgggacg cttgagtatg gtagaggttg gtggaactgc 600 gagtgtagag gtgaaattcg tagatattcg caagaacacc ggtggcgaag gcggccaact 660 ggaccattac tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg 720 tagtccacgc cgtaaacgat gaatgccagc tgttggggtg cttgcaccgc agtagcgcag 780 ctaacgcttt gagcattccg cctgggagag tacggtcgca agattaaaac tcaaaggaat 840 tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgcagaacc 900 ttaccatcct ttgacatggc gtgttaccca gagagatttg gggtccactt cggtggcgcg 960 cacacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020 aacgagcgca acccacgtcc ttagttgcca tcattcagtt gggcactcta gggagactgc 1080 cggtgataag ccgcgaggaa ggtgtggatg acgtcaagtc ctcatggccc ttacgggatg 1140 ggctacacac gtgctacaat ggcggtgaca gtgggacgcg aaggagcgat ctggagcaaa 1200 tccccaaaag ccgtctcagt tcggattgca ctctgcaact cgagtgcatg aaggcggaat 1260 cgctagtaat cgtggatcag catgccacgg tgaatacgtt cccgggcctt gtacacaccg 1320 cccgtcacac catgggagtt ggtcttaccc gacggcgctg cgccaaccgc aaggaggcag 1380 gcgaccacgg tagggtcagc gactggggtg agtcta 1416

Claims (5)

Methylobacterium fujisawaense CBMB 20 strain (KACC-91209P) having a nucleotide sequence of SEQ ID NO: 1 for promoting plant growth. The novel Methylobacterium fujisawaense CBMB 20 strain (KACC-91209P) 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 20 strain (KACC-91209P) 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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