KR101512663B1 - Novel strain of microbes and Ecofriendly fertilizer containing the same - Google Patents

Abstract

The present invention relates to a strain Pseudomonas brassicacearum subsp. neoaurantiaca More specifically, the present invention relates to a strain Pseudomonas , which is a strain that inhibits the growth of plants and inhibits plant growth and accelerates aging, lowers the level of ethylene, and sustains the growth of plants under stress conditions, brassicacearum subsp. neoaurantiaca Investigating the production and activity of ACC deaminase related to BD2-26; Examining the biological performance of the plant hormone; Irradiating tomato root elongation; The present invention has the effect of accelerating the growth of plants. If the strain promoting plant growth is used as a microbial fertilizer for plants under stress, it is possible to protect the crops, And is useful in the crop growing industry.

Description

[0001] The present invention relates to novel microbial strains and microbial preparations for environmentally friendly fertilizers,

The present invention relates to a strain providing plants with ACC deaminase and plant growth promoting hormone involved in promoting plant growth under various conditions.

As heat and drought affect grain growth, grain prices rise, and drought in some areas due to global warming can damage crop production, an effective way to protect crops is needed. When plants are stressed under various conditions of the plant environment, the production of ethylene in plant tissue cells increases (Morgan and Drew, 1997), which inhibits root growth and flowering of plants.

On the other hand, plant growth promoting rhizobacteria (PGPR) promotes plant growth directly or indirectly due to various types of bacteria present in the rhizosphere or in the root (Ahmad et al., 2008).

In a manner that promotes plant growth, there are direct methods of providing plants with growth promoting compounds synthesized from bacteria and indirect growth promoting the reduction or prevention of harmful effects of plant pathogens. An example of indirect growth promotion is the action of 1-aminocyclopropane-1-carboxylate (ACC deaminase), which provides plant growth-promoting hormones to plants, (Glick, 1995). It is possible to decrease the concentration of ethylene in plants.

PGPR is a plant hormone secretion such as auxin, cytokinin, gibberellin, and abscicisic acid (ABA) that contribute to plant growth promotion.

Korean Patent Laid-Open No. 10-2011-0122257 relates to a method for promoting plant growth using microorganisms isolated from a root of Miscanthus sacchariflorus, and relates to a method for promoting plant growth using Tumebacillus sp. sp . ) BE501, Bacillus sp. sp .) BE506, Agrobacterium sp .) BE516 and the genus Lysinibacillus sp . ) BE533 strain produced Auxin and ACC deaminase which promote plant growth and showed remarkable seed germination promoting effect on wheat, wheat, barley, rice, Sudan grass, Chinese cabbage, cucumber and tomato And a growth promoting effect. However, in the above-mentioned Patent Documents, Pseudomonas brassicaearum subunit BD2-26 ( Pseudomonas brassicacearum subsp. neoaurantiaca BD2-26) contributes to promoting the growth of the plant.

Accordingly, it is an object of the present invention to provide a strain which produces an enzyme, ACC deaminase, which reduces stress ethylene, which inhibits plant growth, and produces a plant hormone with plant growth promoting effect.

Another object of the present invention is to provide a microorganism preparation for eco-friendly fertilizer comprising the strain as an active ingredient.

The above object of the present invention can be accomplished by a method for detecting the activity and activity of an ACC deaminase of a strain; Examining the production ability of the plant hormone; Irradiating tomato root elongation; This study was accomplished through the experiment and evaluation of small scale soil cultivation of tomato.

The present invention has the effect of promoting the growth of plants under stress conditions by lowering the level of plant hormone ethylene which inhibits the growth of plants and promotes aging under various conditions. The strain promoting the growth of plants is used for plants under drought stress If used as a microbial fertilizer, it has an excellent effect of protecting crops.

FIG. 1 is a graph showing root elongation at the time of germination of tomato seeds against ACC deaminase and plant hormone-producing strains.
FIG. 2 is a graph showing the dry weight of tomato plants cultivated for 7 days on ACC deaminase and strains having plant hormone production ability.

Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited thereto.

Example 1: Isolation and Identification of Microorganism Strain of the Present Invention

In order to isolate strains, strains were isolated from the rhizosphere of the sand dune plants which are affected by water deficiency and salinity stress. Lxeris repens and Carex kobomugi were collected at Kyunggi - do, and Liriope platyphylla was collected at Gyeongpo beach. The soil attached to the roots of the collected plants was bled off and mixed with physiological saline (0.9% NaCl), diluted and plated on LB agar medium. The subculture was repeated at 30 ° C to isolate the strains pure. Sequence analysis of 16S rRNA gene was performed in Macrogen Co., Ltd. to identify strains BD3-35 and BD2-26, respectively, which were isolated purely from Rhizophora japonica and Tongbori rhizosphere, respectively, and m-2 and m-4 isolated from rhizosphere rhizosphere The entire nucleotide sequence of the obtained 16S rRNA gene was identified using EzTaxon server 2.1 (http: //147.47.212.35.8080/index.jsp). The nucleotide sequence of the gene is described in the Sequence Listing.

The results showed that strains BD3-35 and BD2-26, isolated from Rhizopus japonicus and Tongbori rhizosphere, did not grow in the negative control medium without nitrogen source, but grown in the experimental medium supplemented with ACC as the only nitrogen source. As compared with the control medium, it was found that the strains were cultured using ACC as a nitrogen source. As a result, strains were selected as strains capable of producing ACC deaminase. When comparing their 16S rRNA gene sequences with NCBI-registered strains, BD2-26 was found to be Pseudomonas brassicacearum subsp. neoaurantiaca And 100%, respectively.

The strain identified above was deposited at KACC91820P at BiDi 2-26 (BD2-26) on Jul. 15, 2013 at the National Institute of Agricultural Science and Technology.

Example 2: ACC deaminase Productivity and  Activity investigation

The strains were selected based on their ability to grow using ACC as the sole nitrogen source. 100 μl of 10 mM ACC was applied to the minimal agar medium containing no nitrogen source and used as an experimental group. The addition of 2.0 g / L (NH₄) ₂ SO ₄ as a nitrogen source was considered as a positive control group and the addition of nothing as a negative control group.

Strains were inoculated and cultured at 30 ℃ for 3-14 days. Strains isolated from the negative control group but not grown in the positive control group were selected and tested.

ACC-available strains were inoculated in 7.5 ml of DF medium supplemented with 3 mM of ACC, washed with centrifugation, and then 600 μl of 0.1 M Tris-HCl (pH 8.5) and 30 μl of toluene were added thereto. cell.

ACC deaminase activity was measured by using toluenized cell and protein quantification was measured by Bradford assay in toluenized cells.

The enzyme activity was expressed in terms of the concentration of α-ketobutyrate produced in one hour by the strain.

Example 3: Plant hormone Generation  Research

The abscicisic acid (ABA) and cytokinin production ability of other plant hormones of the present invention strains were examined.

The strains were preincubated in 100 ml of brain heart broth medium (72 h, 30 ° C) and then corrected for OD 600 to 1 using a spectrophotometer.

10 ml of the corrected strain was inoculated in 90 ml of brain heart broth (BHB) medium and cultured in the absence of light (30 ° C, 200 rpm).

After centrifuging the culture, 100 ml of the supernatant was adjusted to pH 2.5, 15 ml of ethyl acetate was added, and the mixture was added to a separatory funnel. The mixture was extracted with an extraction shaker for 15 minutes and the ethyl acetate layer was collected three times ml of ethyl acetate was recovered.

The collected ethyl acetate was evaporated in a vacuum rotary evaporator, dissolved in 5 ml of methanol and filtered through a 0.22 μm oil-soluble filter. Quantitative analysis was performed using a Luna 5μC18 column with a high performance liquid chromatograph (HPLC) and a flow rate of 1 ml / min. ABA, 55% methanol with 0.1 M acetic acid and 70% methanol with cytokinin were used as mobile phases and analyzed at 265 nm and 254 nm wavelength, respectively. All plant hormone production by the strain was expressed as protein value (㎍ / mg protein).

As a result, BD2-26 strain produced cytokinin and showed high productivity of 14.94 μg / mg protein (3.54 μg / mL) and 15.61 μg / mg protein (3.61 μg / mL) on the first day of culture.

Example  4: Tomato root height investigation

In order to investigate the inhibition of ethylene production and promoting root growth, CoCl₂, an ethylene production inhibitor, was treated as a control.

Twenty aliquots of alcohol-sterilized tomato seeds were placed in a sterile glass Petri dish with sterile filter paper, followed by 6 ml of each strain, 6 ml of 2 μM CoCl₂ and 6 ml of sterile distilled water. The root length of germinated tomato seedlings was measured after incubation for 6 days at 30 ℃ in dark condition. The results were analyzed by using ANOVA, SYSTAT ver. 10, 2000, Systat Software, USA) Respectively.

As shown in Fig. 1, BD2-26 showed the greatest increase in root length of germinated tomato seedlings by 161%.

Example  5: Small scale soil cultivation experiment of tomato

The soil was sieved with a 2 mm sieve, and the mixture was mixed so that the ratio of soil and commercial by-product compost was 70:30, and then 250 g of each were contained in three plastic containers each having a diameter of 7.5 cm.

Four tomato seedlings germinated with a root length of less than 1 cm were planted at a depth of 2 cm and cultivated for 7 days in a plant growth chamber under light conditions (12 h, 30 ° C, 6,000 lux) and dark conditions (12 h, 24 ° C) Twenty milliliters of distilled water was added each time to grow 20% of soil moisture.

Experimental results As shown in Figure 2 the present invention Pseudomonas brassicacearum subsp. neoaurantiaca BD3-35 strain was significantly increased by 68% compared to the control group and BD2-26 strain of the present invention having cytokine production ability showed the greatest increase in dry weight to 33% of the control group.

Agricultural Biotechnology Research Institute KACC91820 20130715

<110> Kangwon national university <120> Novel strain of microbes and Ecofriendly fertilizer containing          the same <130> P13-075 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1537 <212> RNA <213> Pseudomonas brassicacearum subsp. neoaurantiaca BD2-26 <400> 1 ggaaggaggg ttgtttgggg cgggtttttt tttttttttt tattctcccg cctagatgac 60 gctggcggca ggcctaacac atgcaagtcg agcggtagag aggtgcttgc acctcttgag 120 agcggcggac gggtgagtaa agcctaggaa tctgcctggt agtgggggat aacgctcgga 180 aacggacgct aataccgcat acgtcctacg ggagaaagca ggggaccttc gggccttgcg 240 ctatcagatg agcctaggtc ggattagcta gttggtgagg taatggctcca ccaaggcgac 300 gatccgtaac tggtctgaga ggatgatcag tcacactgga actgagacac ggtccagact 360 cctacgggag gcagcagtgg ggaatattgg acaatgggcg aaagcctgat ccagccatgc 420 cgcgtgtgtg aagaaggtct tcggattgta aagcacttta agttgggagg aagggcatta 480 acctaatacg ttagtgtttt gacgttaccg acagaataag caccggctaa ctctgtgcca 540 gcagccgcgg taatacagag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcgc 600 gtaggtggtt cgttaagttg gatgtgaaat ccccgggctc aacctgggaa ctgcattcaa 660 aactgtcgag ctagagtatg gtagagggtg gtggaatttc ctgtgtagcg gtgaaatgcg 720 tagatatagg aaggaacacc agtggcgaag gcgaccacct ggactgatac tgacactgag 780 gtgcgaaagc gtgggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat 840 gtcaactagc cgttgggagc cttgagctct tagtggcgca gctaacgcat taagttgacc 900 gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc 960 ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggcc ttgacatcca 1020 atgaactttc cagagatgga ttggtgcctt cgggaacatt gagacaggtg ctgcatggct 1080 gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgt aacgagcgca acccttgtcc 1140 ttagttacca gcacgtaatg gtgggcactc taaggagact gccggtgaca aaccggagga 1200 aggtggggat gacgtcaagt catcatggcc cttacggcct gggctacaca cgtgctacaa 1260 tggtcggtac agagggttgc caagccgcga ggtggagcta atcccacaaa accgatcgta 1320 gtccgggatc gcagtctgca actcgactgc gtgaagtcgg aatcgctagt aatcgcgaat 1380 cagaatgtcg cggtgaatac gttcccgggg ccttgtacac accgcccgtc acaccatggg 1440 gaagtgggtt gcaccagaaa gtagctagtc taaccttcgg gggggacggt taccacggtg 1500 tgattcatga tgggggagag aaggagagag gcaaaaa 1537

Claims (2)

( Pseudomonas brassicacearum subsp. Neoaurantiaca BD2-26) strain having the gene represented by SEQ ID NO: 1 and having the ability to produce ACC deaminase KACC91820P).
A microorganism preparation for eco-friendly fertilizer containing the Pseudomonas brassicaearum subsp. Coli 2-26 strain (KACC91820P) of claim 1 as an active ingredient.

Images