KR20150105548A - Novel microbial agent promoting the growth of plants comprising Arthrobacter woluwensis ED and immobilized body of the same - Google Patents

Abstract

The present invention relates to a microbial agent containing a novel microbe, Arthrobacter woluwensis ED, which has a promoting activity for plant growth, and an algaenate beads immobilized with the same onto an immobilized carrier as active ingredients. According to the present invention, the microbial agent is collected by the following steps: producing immobilizing strains; cultivating the immobilizing strains acquired from the previous step in soil on a small scale and conducting a field test at Paroho Lake; comparing and evaluating a growth of the plants acquired from the previous step; and analyzing a colony of bacteria in a rhizosphere soil for the plant. In addition, the microbial agent has excellent effects of sustaining a survival of the microbial agent around a rooting zone and supplying the same continuously.

Description

[0001] The present invention relates to a novel microbial agent for accelerating plant growth, which comprises a novel strain of Aspergillus wortensis ED and a carrier immobilizing the same,

The present invention relates to a novel microorganism preparation containing Aspartame Wallunensis ED strain having plant growth promoting activity and an alginate bead in which it is immobilized on a carrier for immobilization.

In Korea, about 65% of the annual precipitation is concentrated between June and September, so seasonal variation of annual precipitation is large and 65% of the country is mountainous. For this reason, many multi-purpose dams are installed for the supply of related water, flood control, and power generation. However, the artificial lake formed due to the dam increases the difference between the high water level and the low water level due to the concentrated rainfall and the artificial discharge in the summer, and the vegetation of the aquatic vegetation does not grow due to the large water level difference and the ecosystem of the lake coast is damaged, Et al., 2003). Because of the lack of vegetation, the land is easily eroded and the terrestrial ecosystem and the aquatic ecosystem are cut off, resulting in a decrease in fish and aquatic insects. However, this method requires a lot of restoration cost due to the large scale of the construction, and it is difficult to transport used materials and heavy equipment (Yu, 2010). Therefore, it is necessary to develop a low-cost environmentally friendly method, and the use of microbial fertilizer can be an alternative to the restoration of vegetation (Wu et al . , 2009).

The use of plant growth promoting rhizobacteria (PGPR), which can interact with plants as microbial fertilizers to produce plant hormones, has been attracting attention and has been reported to be effective in promoting plant growth when applied to plants (Segura et al. , 2009). The survival period of PGPR inoculated with plants as microbial fertilizers is affected by temperature, bacteria, species characteristics, soil characteristics and root secretions (Dutta et al . , 2010). One type of PGPR is auxin, gibberellin, and absicic acid (ABA), which are excellent in the ability to dissolve phosphoric acid. This PGPR is inoculated by suspending the bacteria in water and inoculating the plants Have been used frequently. However, this inoculation method has disadvantages that PGPR is sensitive to salt stress, temperature and ultraviolet ray, and it is difficult to clustulate by reaching plant rhizome (Wu et al . , 2012). If the inoculated PGPR can increase colonization and survival in plant rhizosphere, it will be possible to reduce the cost and labor of the restoration of the prairie, and restore more effective vegetation. Therefore, an effective method is needed to increase the survival period of microorganisms and to inoculate the microorganisms in water for continuous supply, and microorganism immobilization method can be applied to them.

Microbial immobilization has been improved to provide nutrients, to prevent soil drying, and to provide a long and slow supply of immobilized microorganisms (Bashan, 1998; Kim et al . , 2012). Immunization with PGPR has been shown to be effective in promoting plant growth in wheat and tomato (Bashan, 1998) and helping to colonize the plant rhizosphere by meeting the number of bacteria required for PGPR clustering (Heijnen and Van Veen, 1991). Carriers used for microorganism immobilization include sodium alginate, polyacrylamide, and xanthan gum. Among them, sodium alginate, which is non-toxic, biodegradable and simple to manipulate, has been widely used (Bashan, 1998). Alginate is a chain-like polymer in which D-mannuronic acid and - (1,4) are linked by L-gluronic acid. Sodium alginate is attached as the Na ⁺ in the chain between the Na ⁺ are each substituted with a Ca ^{2 +} CaCl _2. Since Ca ^{2 +} is a bivalent metal ion, it forms a porous Ca ^{2 +} -alginate polymer by binding to two sodium alginate chains (Haug, 1959). Immunization with PGPR in alginate beads has been reported to maintain PGPR survival in rhizosphere and to provide sustained availability (Bashan, 1986).

As a method for immobilizing microbial biofilm formation inhibitor using a hydrogel as a fluidized bed carrier and a water treatment apparatus for a membrane using the same, disclosed in Korean Patent No. 10-1270906, there is disclosed a method for producing immobilized microorganisms using a prepolymer and a cross- Korean Patent Laid-Open Publication No. 10-2006-0043555 discloses a carrier for immobilizing methanotrophic bacteria and a method for removing methane using the same, in Japanese Laid-Open Patent Publication No. 10-2013-0120309. However, none of the above patent publications discloses or suggests any microorganism preparation in which the plant growth promoting active microorganism is immobilized on the immobilizing carrier.

Therefore, it is an object of the present invention to provide a method for promoting the growth of plants, which comprises administering an effective amount of Arthrobacter woluwensis ED strain (KCTC 12011BP).

It is another object of the present invention to provide an immobilized strain in which a strain according to the present invention is immobilized on an immobilizing carrier.

It is still another object of the present invention to provide a microorganism preparation for promoting plant growth, comprising the strain or immobilized strain according to the present invention as an active ingredient.

The above object of the present invention can be achieved by a method for producing an immobilized strain, Performing small-scale soil cultivation and Faro field trial of the immobilized strains obtained in the above step; Comparing and evaluating the growth of the plant obtained in the step; And analyzing bacterial populations of the plant rhizosphere soil in the above step.

The present invention has an excellent effect of maintaining and continuously supplying the survival of the microorganism preparation in the rhizosphere.

FIG. 1 is a graph showing the stem length (A) and the root length (B) of a tomato treated with the rhizobia bacterium A. woluwensis ED strain according to the present invention.
FIG. 2 is a graph showing wet weight (C) and dry weight (D) of tomatoes treated with the rhizobia bacterium A. woluwensis ED strain according to the present invention.
FIG. 3 is a graph showing the stem length (A) and the root length (B) of the microspheres treated with the rhizobia bacterium A. woluwensis ED strain according to the present invention.
FIG. 4 is a graph showing (C) wet weight and (D) dry weight of the mustard seedlings treated with the rhizobia bacterium A. woluwensis ED strain according to the present invention.
FIG. 5 is a photograph showing the results of DGGE analysis of DNA extracted from a cultivated soil sample and a culture medium of a small scale cultivation experiment. ED: A. woluwensis ED strain, CTL: control, SI: culture medium, II: immobilized strain, AC: alginate bead.
6 is a graph showing band intensities of DGGE analysis results.
FIG. 7 is a photograph showing the results of DGGE analysis of DNA extracted from a rhizosphere soil sample and a culture medium of a strain of the Faroe leech.
Figure 8 is a dendrogram showing the similarity of the strain suspension and immobilized strain inoculum.
Figure 9 is a Dendrogram showing the degree of similarity after 3 weeks of inoculation.
10 is a Dendrogram showing the degree of similarity after 6 weeks of inoculation.
Figure 11 is a Dendrogram showing the degree of similarity after 9 weeks of inoculation.

Hereinafter, the present invention will be described in detail with reference to examples.

Published sample

Prepare YEG liquid medium (yeast extract 1%, glucose 10%, pH 6.8) diluted ^10-1 times with distilled water and pH adjusted to 6.8. 100 mL of YEG liquid medium was added to a 250 mL flask, and A. woluwensis ED strain was inoculated, followed by shaking culture at 30 DEG C and 150 rpm for 48 hours. The cultured strains were separated by centrifugation (4 ° C, 3400g, 20 minutes), and the number of bacteria was measured by a hemocytometer, and the cells were inoculated at 1 × 10 ⁶ cells g ^-1 in the soil. The A. woluwensis ED strain was deposited by the present inventor on Aug. 26, 2011 (KCTC 12011BP) to Korea Institute of Bioscience, which is a microorganism international depository institution.

Example : Preparation of immobilized strain

The alginate bead preparation method of Bashan (1986) was modified and used according to the characteristics of the strain. 20 g of sodium alginate (Junsei, Japan) was dissolved in 500 mL of distilled water, and distilled water was added thereto. The mixture was stirred in a magnetic stirrer having a final volume of 1 L for about 30 minutes. Then, a high pressure steam sterilizer (JSAC- Korea) and sterilized under high pressure at 121 ° C and 14.5 Psi for 15 minutes. Prepare 2% sodium alginate solution to use 500 mL per 1 × 1 m ² , dissolve 30 g of calcium chloride in 1 L of distilled water, add distilled water to final volume of 2 L, sterilize by high pressure psi, 15 minutes) was prepared by cooling in a refrigerator (4 ° C). By centrifuging the prepared bacteria enables soil 1 × 10 ⁶ cells per g 1 can be inoculated (4 ℃, 3400g, 20 minutes) to remove the supernatant and washed twice with distilled water was mixed with 2% sodium alginate solution. Peristaltic pump the Latex tube connected to the 22G needle is easy load®-1 pump heads connected (7553-70, Cole Parmer Instrument Co., USA) and then the device, a sterile 1.5% was previously cooled to strain -alginate mixture CaCl ₂ And the mixture was stirred at room temperature for 2 hours with a magnetic stirrer and then hardened. The prepared alginate beads were washed twice with distilled water and then stored in a refrigerator (4 ° C).

Experimental Example  1: Small scale soil cultivation experiment

Soil samples were collected from the Palo Hoang Reservoir in Hwacheon - gun, Gangwon - do. The collected soil was filtered with a 5 mm sieve, and then 250 g of the soil was placed in a plastic pot having a diameter of about 7 cm and a height of about 10 cm. Tomatoes ( Lycopersicon esculentum) 70% sterile 5 minutes the surface in ethanol, placed into a sterile distilled water 5 ml and tomato seeds on a petri dish into autoclaved a cotton wool of 3 mm thick light conditions (30 ℃ for 3 days plant growth chamber, 3250 lux seed) 10 hours and dark condition (24 ℃) for 14 hours. Potted germinated tomatoes were sown in 3 pots in 24 pots and 6 pots were divided into control group, strain suspension inoculation group, immobilized strain inoculation group and control alginate bead group. The inoculum of each test group was inoculated at the same dose of 1 × 10 ⁶ cells g ^{- 1} . Control alginate bead was prepared and sprayed with the same amount of sodium alginate as the immobilized strain. Then, the cells were cultured for 30 days in a plant growth chamber under light condition (30 ° C, 3250 lux) for 10 hours and dark condition (24 ° C) for 14 hours.

The stem length, root length, wet weight, and dry weight of tomatoes were significantly increased by 36.2, 59, 51.1 and 37.5% in the control group, respectively. In the control group, The experimental groups were significantly increased by 42, 67.4, 62.5 and 60.4%, respectively. Therefore, immobilized strain inoculation group increased by 6, 8, 11 and 23%, respectively, compared with the strain suspension inoculation group. In addition, the control group of strain-free control alginate bead was increased by 3.86, 2.6, 13.7 and 17.7%, respectively, but it was not significantly influenced by the absence of significance (Figs.

Experimental Example  2: Faro field experiment

It is an artificial lake formed by Hwacheon Dam constructed in 1939, located in Gwanmari, Gangdong-myeon, Hwacheon-gun, Gangwon-do. The coast of Paro Island is formed by a wide anvil with a slope of 30 °. In order to prepare the experimental area, a large stone of a size large enough to affect the results was filtered out, and a 1 m × 1 m square was added to the control group, the suspension suspension inoculation group and the immobilized strain inoculation group, A square was installed. The planting of the experimental plants was carried out with 25 seedlings per each square. The weights of 1 m × 1 m × 20 cm (width, height, height) were measured and weighed about 100 kg. The inoculum of each experimental group was inoculated at 1 × 10 ⁶ cells g ^{- 1} at 3 - week intervals starting from the first microinjection on April 6, 2013. In the control group, 4 L of water was given per square area using the lyophilus of Paroho, and the strain suspension inoculation group gave the same amount of water suspended in the water of the bacteria. Immobilized strain Inoculum The immobilized strain was also immersed in water and given equal amounts of water.

The root length, root length, wet weight, and dry weight of the leaves were increased by 7, 10.8, 30.7 and 12.6%, respectively, in the control group. In the control group, 36.3%, 32.5%, 64% and 48.9% of immobilized strain inoculation groups were significantly increased, respectively. Therefore, immobilized strain inoculation group increased significantly in all items except wet weight item by 30, 21, 33 and 36%, respectively (Fig. 3, 4).

Experimental Example  3: Bacterial community survey of rhizobacteria according to the present invention

G-DNA was extracted from the rhizosphere soil collected during the small-scale soil cultivation and the Faroho field experiment according to the manual of Powersoil DNA isolation kit (MO BIO Laboratories, Inc., USA) and the culture broth of A. woluwensis ED in YEG medium G-DNA was extracted according to the manual of G-spin ^TM genomic DNA extraction for bacteria (Intron Biotechnology, Korea).

The g-DNA extracted from the cultivated rhizosphere soil was subjected to PCR to amplify the DNA because the amount of DNA was small for DGGE analysis. For amplification of 16s rDNA region, forward primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3 ') and reverse primer 515R (5'-ACCGCGGCTGCTGGCAC-3') were used for the first PCR, forward primer F352TA -CGCCCGCCGCGCGCGGGGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGC-3 ') and reverse primer 515R such as primary PCR were used. The first PCR was performed using 2 μL of DNA template, 1 μL of 27F, 1 μL of 515R, 2 μL of 10 × taq polymerase buffer, 2.5 μL of 2.5 mM dNTP, and 5 μL of Ex-taq polymerase (Takara Shuzo Co., Japan) was added and the final volume was adjusted to 25 μL by adding tertiary distilled water. In the second PCR, 1 μL of the first PCR product was diluted to 10 ^-2 , and the remaining PCR products were mixed with the same amount of the first PCR, and the final volume was adjusted to 25 μL with the third distilled water. The primary PCR conditions consisted of initial denaturation (94 ° C, 5 min), denaturation (94 ° C, 30 sec), annealing (58 ° C, 30 sec) and extension (72 ° C, 40 sec) Followed by final extension (72 ° C, 3 min). Subsequently, the secondary PCR was modified by partially modifying the primary PCR conditions, modifying the extension step of the cycle step to 30 seconds, repeating this step for 40 cycles, and then performing final extension (72 ° C, 5 minutes). In order to confirm the amplification of the DNA, PCR product and 6x DNA loading dye were mixed in a ratio of 5: 1, and the mixture was placed in 1% agarose gel. The PCR product was electrophoresed in a PTC DNA Engine system (Bio-Rad Laboratories Inc., USA) DNA amplification was confirmed by electrophoresis and this product was used in DGGE.

The denaturing solution used for DGGE gel concentration gradient was prepared by mixing 17.5 mL of 40% Acrylamide / Bis (37.5: 1) and 1 mL of 50x TAE buffer, then adding formamide and urea, adding 3 rd distilled water, Make it 50 mL. The 43% denaturing solution was prepared by adding 8.6 mL of formamide and 9.03 g of urea and adding 12.6 mL and 13.23 g of 63% denaturing solution, respectively. 150 μL of APS (2 g of ammonium persulfate, 10 mL of tertiary distilled water) and 10 μL of N, N, N, N-tetramethylethylene diamine (TEMED: Sigma Chemical Co., USA) were mixed with 15 mL of each denaturing solution, Concentration gradient to form polyacrylamide gels. Then, each well of the gel was washed twice with 3 times distilled water, washed once with 0.5x TAE buffer, and 15 μl of the soil g-DNA amplified by PCR was mixed with 15 μl of 2x loading dye in a ratio of 1: 1 Samples stained with 30 μL were injected into each well and placed in a DGGE instrument DCode ^™ universal mutation detection system (Bio-Rad, USA). The temperature of the 0.5 x TAE buffer in the device was maintained at 64 ° C, pre-run at 20 V for the first 30 minutes and then electrophoresed at 60 V for about 24 hours. After electrophoresis, gel was added to a mixture of 10 μL of ethidium bromide and 30 mL of 0.5 × TAE buffer. Third distilled water was added to a final volume of 100 mL, followed by stirring for 20 minutes. After dyeing, the cells were washed twice with distilled water, and gel was added to 100 mL of distilled water for decoloration for 20 minutes. Using a discoloration after UV transilluminator (SL-20 High Performance DNA Image Visualizer TM, Seoulin Scientific Co., Korea) into the gel Gel Logic 100 imaging system and molecular imaging software version 4.0 (Eastman Kodak Co., USA) The patterns and intensities of the bands were analyzed.

The results of DGGE (Fig. 5) using g-DNA extracted from the rhizosphere soil samples and the g-DNA extracted from the A. woluwensis ED strain culture medium in small scale soil cultivation (Fig. 5) showed that the A. woluwensis ED strain was inoculated into the rhizosphere soil microbial community . ≪ / RTI > The band intensity of A. woluwensis ED strains in each bacterial community was compared and plotted (FIG. 6). As a result, immediately after inoculation, the strains showed the highest band intensity and decreased gradually, whereas the strains Intensity increased rapidly for 1 week after inoculation and then maintained high band intensity for more than 3 weeks.

DGGE was performed using g-DNA extracted from rhizosphere soil samples and g-DNA extracted from the culture of A. woluwensis ED strain during the field restoration of the Paroho-Hoan wild field. As a result, A. woluwensis ED strain And remained in the soil bacterial community (Fig. 7). Immediately after inoculation, the strains showed the highest band intensities, indicating that strains were applied. In the non - inoculated control group, A. woluwensis ED strains were found to be present in trace amounts. Three weeks after the inoculation, a small amount of A. woluwensis ED strain in the non-inoculated control group was thought to have formed a bacterial community by interacting with the pinworm. The band intensities of the strain suspension inoculation group were the highest immediately after inoculation, but after 3 weeks, the band intensities were lower than in the immobilization strain (Table 1).

Experimental Example  4: Cluster analysis of rhizobacteria according to the present invention

Soil samples were collected six times during the 15 - week period during the Paroho field experiment and analyzed by DGGE. The results of DGGE analysis were used to measure the similarity of each bacterial community using Pearson correlation and unweighted arithmetic, and dendrograms were drawn.

As shown in FIG. 8 to FIG. 13, the similarity of the strain suspension inoculated with the strains was 95, 77, 86, 92, 85 and 80%, which was 85.8% The similarity of the control group was 87, 75, 73, 75, 80 and 68%, which showed an average similarity of 76.3%.

Korea Biotechnology Research Institute KCTC12011BP 20110826

<110> KNU-Industry Cooperation Foundation <120> Microbial agent promoting the growth of the plants          Arthrobacter sp. ED or immobilized body of the same <130> P5476 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1398 <212> RNA <213> Arthrobacter woluwensis <400> 1 taccatgcag tcgaacgatg aagcctagct tgctgggtgg attagtggcg aacgggtgag 60 taacacgtga gtaacctgcc cttgactctg ggataagcct gggaaactgg gtctaatacc 120 ggatacgacc attgcccgca tgggttggtg gtggaaagct tttgtggttt tggatggact 180 cgcggcctat cagcttgttg gtgaggtaat ggctcaccaa ggcgacgacg ggtagccggc 240 ctgagagggt gaccggccac actgggactg agacacggcc cagactccta cgggaggcag 300 cagtggggaa tattgcacaa tgggcgaaag cctgatgcag cgacgccgcg tgagggatga 360 cggccttcgg gttgtaaacc tctttcagta gggaagaagc gaaagtgacg gtacctgcag 420 aagaagcgcc ggctaactac gtgccagcag ccgcggtaat acgtagggcg caagcgttat 480 ccggaattat tgggcgtaaa gagctcgtag gcggtttgtc gcgtctgctg tgaaaggcca 540 gggctcaacc ctggttctgc agtgggtacg ggcagacttg agtgatgtag gggagactgg 600 aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccgatg gcgaaggcag 660 gtctctgggc attaactgac gctgaggagc gaaagcatgg ggagcgaaca ggattagata 720 cccctggtag tccatgccgt aaacgttggg cactaggtgt gggggacatt ccacgttttc 780 cgcgccgtag ctaacgcatt aagtgccccg cctggggagt acggccgcaa ggctaaaact 840 caaaggaatt gacgggggcc cgcacaagcg gcggagcatg cggattaatt cgatgcaacg 900 cgaagaacct taccaaggct tgacatggat cagatcgcat cagagatggt gtttcccttc 960 ggggctggtt cacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1020 agtcccgcaa cgagcgcaac cctcgttcca tgttgccagc gcgtaatggc ggggactcat 1080 gggagactgc cggggtcaac tcggaggaag gtggggacga cgtcaaatca tcatgcccct 1140 tatgtcttgg gcttcacgca tgctacaatg gccggtacaa agggttgcga tactgtgagg 1200 tggagctaat cccaaaaagc cggtctcagt tcggattggg gtctgcaact cgaccccatg 1260 aagttggagt cgctagtaat cgcagatcag caacgctgcg gtgaatacgt tcccgggcct 1320 tgtacacacc gcccgtcaag tcacgaaagt tggtaacacc cgaagccggt ggcctaaccc 1380 cttgtgggag ggagctgt 1398

Claims (4)

A promoter for the growth of plants is Arthrobacter having the rRNA nucleotide sequence shown in SEQ ID NO: 1 ( Arthrobacter woluwensis ED strain (KCTC 12011BP). The method of claim 1, wherein Arthrobacter woluwensis) ED immobilized strains were the alginate bead-immobilized carrier to secure the strain as an effective ingredient. A microorganism preparation for promoting plant growth, comprising the Aspergillus wortensis strain of claim 1 or the immobilized strain of claim 2 as an active ingredient. A method for promoting plant growth, characterized in that the microorganism preparation containing the strain of Aspergillus wallulensis of claim 3 or an immobilized strain thereof is cultivated by spraying with soil or inoculating the plant seed.

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