KR101243349B1 - Pseudomonas aurantiaca IB5-10 having environmental stresses resistance and plant growth promotion, microbial agent containing the same, and method for recovering of coastal sand dune plants - Google Patents

Abstract

The present invention relates to Pseudomonas aurantiaca IB5-10 having an environmental stress relaxation and color growth promoting function, and more specifically, a Korean agricultural microorganism having an environmental stress relaxation and plant growth promoting function of sand dunes, isolated from the root zone of sand dunes. It relates to strain Pseudomonas aurantiaca IB5-10 deposited as KACC 91561P in the repository.
According to the present invention, a multifunctional coastal species having the effects of dominance, fixation ability, growth promoting ability, pathogenic activity, environmental stress resistance induction, etc. among the old plant microspheres living in the root zone of coastal sand dunes and coexisting with sand dunes Sand dune PGPR can be used to develop microbial agents that can be used to restore sand dunes. As confirmed in the embodiment of the present invention, when the developed microbial agent was treated to the destroyed coastal sand dunes, not only the growth of coastal sand dunes but also the germination rate increased. Therefore, the restoration of the coastal sand dunes and ecosystem restoration using the microbial preparation of the present invention is expected.

Description

Pseudomonas aurantiaca IB5-10 having environmental stresses resistance and plant growth promotion, microbial agent containing the same, and method for recovering of coastal sand dune plants}

The present invention Pseudomonas having a function of environmental stress relief and plant growth promotion The strain Pseudomonas deposited as KACC 91561P in the Korea Agricultural Microbiological Resources Center, which has a function of alleviating environmental stress and promoting plant growth of aquatic plants, isolated from the rhizosphere of aquatic plants, more specifically, aurantiaca IB5-10. aurantiaca Relates to IB5-10.

Coastal sand dunes are a boundary between marine and terrestrial ecosystems and play a very important role as coastal protection and habitats for dune-specific vegetation. However, due to the poor development and pollution of the dune area, it has been damaged to the extent that it is difficult to find the original form, and its seriousness is recognized not only in Korea but also all over the world. The restoration and preservation of the sand dune ecosystem can be accomplished by the installation of facilities to prevent erosion, to create a sedimentary environment, and to artificially cover the ground.However, the restoration of coastal sand dunes through ecological restoration of damaged dune vegetation is the best in terms of the natural ecosystem. It will be eco-friendly and economical. Indeed, developed countries, including the United States and Europe, use the restoration of dune vegetation most frequently as a method of coastal dunes. The dune vegetation, which occupies most of the dune vegetation, has to adapt to the harsh environment such as drying and high salt and various stresses. Factors are greatly affected by seed germination and growth of sand dunes. In addition, the factors that control the growth of plants to plants exposed to environmental stress, such as sand dunes, are plant growth regulators, namely plant hormones. The supply of growth regulators to plant growth is dependent on the amount of endogenous hormones and metabolism, Has a lot of influence on the reproduction and growth of plants. The dune plants produce environmental stress-reducing substances such as plant growth regulators such as ABA (abscisic acid) and JA (jasmonic acid) or levan for growth and breeding under adverse stress conditions. By interacting with microorganisms, it overcomes environmental stresses such as drying and high salt.

On the other hand, sand dunes, which occupy most of the dune vegetation, have to live in the harsh environment such as dryness and high inflammation and various stresses, and their interaction with the root zone microorganisms that coexist and coexist in the root zone for their growth is essential. need. Therefore, the utilization and restoration of these rhizosphere microorganisms should be used for the proliferation and expansion of disappearing glomerular vegetation. There is almost no attempt to utilize microorganisms to restore such sand dunes. However, in crop cultivation and horticulture, many studies have been conducted using plant growth promoting rhizobacteria (PGPR) having plant growth promoting ability and disease prevention ability. Plant growth promoting myobacteria (PGPR), which promotes plant growth, live in the root zone of plants and induce growth-promoting effects by plant growth-promoting hormones, production of various plant growth-regulating hormones, systemic acquisition resistance (SAR) and induction systemic resistance. Induction of defense mechanisms of plants such as ISR and control of phytopathogens. PGPR can be classified into biological control PGPR, which indirectly affects plant growth by inhibiting phytopathogens and promoting plant growth, promoting seed germination, and increasing crop productivity. Microorganisms or microbial pesticides using such PGPR have been developed, and are already widely applied in agriculture or horticultural sites. In particular, the eco-friendly agriculture of green growth is recognized as the trend of the current trend, and the microbial farming method is being implemented as the most representative eco-friendly agriculture. Sand dune plants, such as economic crops and horticultural crops, are considered to be useful for the growth of sand dunes if the above PGPR is used.

Accordingly, the present inventors have made a thorough research to overcome the problems of the prior art, as a result of the advantages, settlement ability, growth promoting ability, disease control ability of the dune vegetation root zone microorganisms living in the root zone of coastal sand dunes and symbiotic with old plants We developed microorganisms that can be used to restore sand dunes by using multifunctional coastal sand dunes PGPR, which have the ability to induce environmental stress resistance and phosphate solubility, and settle in the root zone of sand dunes even in the very disadvantageous growth environment of coastal dunes. A stable formulation method that can be stored and stored for a long time was developed and applied to the native plants in coastal sand dunes. In other words, by developing and formulating a stable formulation method of multi-functional PGPR for the first time in Korea, it lays the groundwork for applied research on the storage and stability of eco-friendly microorganisms and ultimately disappears by using the PGPR of sand dunes. We tried to preserve the devastated sand dune ecology of Korea by restoring.

Therefore, the main object of the present invention is a novel microorganism Pseudomonas having a function of alleviating environmental stress and promoting plant growth. to provide aurantiaca IB5-10.

Another object of the present invention is the novel microorganism Pseudomonas It is to provide a microbial agent comprising aurantiaca IB5-10.

Still another object of the present invention is to provide a method for restoring a glomerular plant using the microbial agent.

According to an aspect of the present invention, the present invention is a strain Pseudomonas aurantiaca IB5-10 deposited as KACC 91561P in Korea Agricultural Microbiological Resources Center, which has a function of alleviating environmental stress and promoting plant growth of sand dunes, isolated from the root zone of sand dunes. To provide.

The strain Pseudomonas of the present invention aurantiaca IB5-10 is characterized in that it comprises 16S rDNA having a nucleotide sequence of SEQ ID NO: 1.

The strain Pseudomonas of the present invention aurantiaca IB5-10 is characterized by having the ability to produce environmental stress relief hormones ABA and JA, and environmental stress relief substance Levan.

ABA is a plant hormone that reacts to environmental stresses such as drying or high salts, and is a plant hormone that induces resistance to plants by controlling the pore opening and closing of plant leaves and invading pathogens, and JA is generally produced in plants that are higher plants. As a plant hormone, it is known as a derivative that induces resistance to plants in response to invasion of pathogens, mechanical injuries, and various environmental stresses.It protects plants by maturation of fruits, water production, root growth, and microbial infection. Involved in the system. Levan is a kind of fructan, and microbial levan regulates seed germination and storage capacity in plants and relieves drought, low temperature and salt stress. It also plays a role in the storage of major nitrogen sources in plants. Due to the nature of coastal sand dunes, it is absolutely necessary for sand dunes that are exposed to large amounts of water evaporation due to solar heat, extreme temperature difference between day and night and malnutrition. In a specific embodiment of the present invention, it was confirmed that the strain Pseudomonas aurantiaca IB5-10 of the present invention produces the above three kinds of substances. Therefore, the strain Pseudomonas of the present invention If aurantiaca IB5-10 is applied in sand dunes, it will be able to improve the coastal vegetation growth by reducing water loss and resistance to pathogens due to pore opening and closing control of sand dunes. It will contribute greatly to the resistance to stress caused by various exposed environmental, mechanical, and pathogens, and also to reduce various environmental stresses and nutrients such as nitrogen source in coastal sand dunes which are located in the environment with rapid temperature difference and lack of water and nutrients. It will be very effective in restoring sand dunes by supplying them.

According to another aspect of the present invention, the present invention provides a microbial agent containing Pseudomonas aurantiaca IB5-10 as an active ingredient.

The microbial agent of the present invention is characterized by increasing the drying and salt stress resistance of sand dunes, and has the effect of promoting the growth of sand dunes.

Specifically Pseudomonas according to the present invention The microbial preparation containing aurantiaca IB5-10, as confirmed in the examples of the present invention, when the stress was applied to the glomerular plants, survival of the glomerular plants continued even after 50 days of the dry stress, as well as drying and Survival of 80% was observed even with salt stress. This is Pseudomonas It can be interpreted that aurantiaca IB5-10 is resistant to environmental stress in sand dunes which are always exposed to stress. Therefore, by having resistance to sand dunes, the survival rate of sand dunes in the dry and high salt condition is improved, which is expected to have a great effect on the restoration of the eco-friendly ecosystem of the destroyed coastal dunes.

The microbial preparation of the present invention is characterized in that the culture solution of Pseudomonas aurantiaca IB5-10 is mixed with an inorganic carrier.

The culture medium is Pseudomonas aurantiaca IB5-10 with Molasses 15-30 g / L, Corn steep liquor 15-30 g / L, Ammonium sulfate 5-15 g / L, Monopotassium phosphate 4-7 g / L, Dipotassium phosphate 3-5 g / L, Magnesium sulfate 2-4 g / L, Manganese sulfate 0.3-0.7 g / L, Antifoam ( Antiforming agent) It is preferably cultured in a medium containing 0.5-0.8 g / L.

In addition, the inorganic carrier is a zeolite composed of AI ₂ O ₃ , SiO ₂ , Fe ₂ O ₃ , CaO, MgO, K ₂ O, Na ₂ O, SO ₃ , TiO ₂ , and LOI, SiO ₂ , AI ₂ Ilite consisting of O _3, Fe ₂ O ₃ , CaO, MgO, K ₂ O, Na ₂ O, P ₂ O ₅ , TiO ₂ , and LOI, or SiO _2, Al ₂ O _3, Na ₂ O , CaO, Fe ₂ O _3, K ₂ O, TiO _{2 ,} and It is characterized in that the H1800C composed of MgO. The inorganic carriers were selected for high stability formulation, and actual Pseudomonas When measuring the storage stability of each type of inorganic carrier of aurantiaca IB5-10, the cell density was maintained at 10 ⁹ CFU / ml for 18 weeks in zeolite during high temperature storage and 10 ⁶ CFU / ml in H1800C. In the low temperature storage, the cell density of both zeolite and H1800C remained almost unchanged.

According to another aspect of the present invention, there is provided a method for restoring a glomerular plant, comprising administering the microbial agent to the glomerular plant.

Administration of the present invention to the glomerular plant is characterized in that the microbial agent is diluted with water and then treated with a spray method.

Restoration of the glomerular plant of the present invention is characterized in that the drying and salt stress resistance is increased, the growth of the glomerular plant is promoted.

Pseudomonas as in the embodiment of the present invention When the microbial preparations of the present invention comprising aurantiaca IB5-10 were administered to the glomerulus plants, it was confirmed that the stem length, root length, number of rootlets, and number of leaflets of the glomeruli increased compared to the untreated control group. Therefore, the microbial agent developed in the present invention has an excellent effect on promoting the growth of sand dunes.

In addition, as a result of performing the sand dunes restoration experiment in the actual coastal sand dunes, the germination rate of sand dunes in the coastal sand dunes treated with the microbial agent was 36.6% compared to the untreated control (0% germination rate). Therefore, if the microbial agent containing Pseudomonas aurantiaca IB5-10 of the present invention is treated to the destroyed coastal sand dunes, it will be a great help in promoting the growth and coastal germination of coastal sand dunes for coastal sand restoration.

In addition, the four-sided plant is characterized in that it is a lotus flower, a magpie swimming or a barley barley.

As described above, according to the present invention, among the musculoskeletal microorganisms inhabiting the rhizosphere of coastal sand dunes and coexisting with sand dunes, the advantages such as dominance, fixation capacity, growth promoting ability, pathogenic ability, and environmental stress resistance inducing ability At the same time, multifunctional coastal sand dune PGPR can be used to develop microbial agents that can be used for restoration of sand dunes. As confirmed in the embodiment of the present invention, when the developed microbial agent was treated to the destroyed coastal sand dunes, not only the growth of coastal sand dunes but also the germination rate increased. Therefore, the restoration of the coastal sand dunes and ecosystem restoration using the microbial preparation of the present invention is expected.

1 is a schematic diagram of a spectrophotometric method for quantifying gibberillin.
2 is a result of measuring gibberillin production capacity.
3 is a schematic diagram of a purification method for HPLC analysis of environmental stress relaxation hormone.
Figure 4 shows the results of HPLC analysis of ABA produced by Pseudomonas aurantiaca IB5-10.
Figure 5 shows the results of HPLC analysis of JA produced by Pseudomonas aurantiaca IB5-10.
Figure 6 is the result of measuring the production capacity of levan Pseudomonas aurantiaca IB5-10 by incubation time (Lane 1, 2% glucose + 2% sucrose + 2% fructose + 1% levan; Lane 2, 2% sucrose; Lane 3, 2% glucose; Lane 4, 2% fructose; Lane 5, 0.1% levan; Lane 6, 0.5% levan; Lane 7, 1% levan; Lane 8, YPC medium; Lane 9, IB5-10 3hr; Lane 10, IB5 -10 6hr; Lane 11, IB5-10 9hr; Lane 12, IB5-10 12hr; Lane 13, IB5-10 15hr; Lane 14, IB5-10 18hr; Lane 15, IB5-10 21hr; Lane 16, IB5-10 24 hr; Lane 25, E. coli 24 hr).
Figure 7 shows the time-phase HPLC analysis of the levan produced by Pseudomonas aurantiaca IB5-10.
Figure 8 shows the results of the dry stress resistance induction effect (30 days) for the flower.
Figure 9 shows the results of the dry stress resistance induction effect (50 days) for the flower.
Figure 10 is a graph of the results of the dry stress resistance induction effect on the flower.
Figure 11 shows the results of the drying and salt stress resistance induction effect on clasp barley.
12 is a graph of the results of the dry stress resistance induction effect on barley.
Figure 13 shows the results of the measurement of cell production according to the type of optimal carbon source for mass culture.
Figure 14 shows the results of the measurement of cell production according to the type of optimal nitrogen source for mass culture.
Figure 15 shows the results of measuring the effect of the commercial medium for mass culture.
16 P Pseudomonas Stability results for the types of inorganic carriers of aurantiaca IB5-10 are shown.
Figure 17 shows a prototype of a microbial agent for promoting glomerular plant growth.
Figure 18 shows the stability results during high temperature storage of microbial preparations for promoting sand dunes plant growth.
Figure 19 shows the stability results during low temperature storage of microbial preparations for promoting glomerular plant growth.
Figure 20 shows the results of the growth promoting effect on the seaweed flower using a microbial preparation.
Figure 21 shows the results of the growth promoting effect on the magpie swimming using the microbial agent.
Figure 22 shows the results of the growth promoting effect on barley bark using a microbial preparation.
Fig. 23 shows the installation of a sowing flower planting area and a rectangle for restoring sand dunes.
Fig. 24 is a picture of germinated germint flowers of an untreated group.
25 is Pseudomonas Seed germination of a microbial preparation containing aurantiaca IB5-10.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  One. Coastal dunes PGPR Separation of

For the isolation of PGPR strains with coastal sand plant growth potential, 11 species of sand dunes (Indigo plant, mudgrass, mudgrass, mud pea, Bodhisattva, mudflats, mud barley, turnip) Soil samples were collected from the root zones of trees, sea breezes, mite flowers, moths, and barley. As a culture medium, 0.1, 0.5, 1 LB (Becton Dickinson, USA) agar medium containing 3% NaCl was used in consideration of high nutrient deficiency and high salinity due to the characteristics of coastal sand dunes. Strain isolation was suspended 1 g of soil samples in 9 ml of physiological saline (0.85% NaCl), diluting step and smeared in each medium, and incubated until colonies were formed at 30 ℃.

As a result of the culture, as shown in Table 1, 1,330 bacteria and 58 actinomycetes could be isolated, and many bacteria could be separated from the root zones of the ulmuflower, mudgrass, Gigi-ri-cho, and mudflats. However, actinomycetes could not be separated more than bacteria. In addition, samples were separated by dividing into coastal military units and other dune areas where the public had little access, and thought that more root zone microorganisms could be separated from the dune areas within the military units. There was a difference in numbers, and the area was not significantly affected.

Table 1. Isolation Result of Bacteria and Actinomycetes in Coastal Sand Dunes

Example  2. Coastal dunes PGPR  Selection

2-1. Auxin Productivity Coastal dunes PGPR  Selection

The Salkowski test [Kim, 2004] was performed to confirm the production capacity of auxin, one of the main plant growth promoting hormones, against 1,330 rhizosphere bacteria and 58 actinomycetes isolated in Example 1. The isolates were isolated at King's B broth (L-tryptophan 0.1%, peptone No. 3 2%, K ₂ HPO ₄ 0.15%, MgSO ₄ 7H ₂ O 0.15%, glycerol 1.5%, pH 7.0) at 28 ° C, 200 rpm. After culturing for 3 days, the strain culture solution was centrifuged at 8000 rpm for 10 minutes. Thereafter, the culture supernatant and Salkowski reagent (27.6 mM FeCl ₃ , 6.6 MH ₂ SO ₄ ) were mixed at 1: 2 (v / v) and reacted for 30 minutes at room temperature under dark conditions. The absorbance of the reaction was measured at 535 nm, and a standard curve was prepared using IAA (indole-3-acetic acid, Sigma, USA) as a standard [Jung et al., 2006].

As a result of the measurement, as shown in Table 2, auxin production capacity was confirmed in 23 kinds of rhizosphere bacteria, and actinomycetes could not be confirmed. All isolates identified as auxin-producing strains were coastal dune PGPRs capable of producing high concentrations of auxin compared to the standard IAA. In addition, the auxin production capacity of these coastal sand dunes PGPR is considered to be of great value in promoting the production of sand dunes in the restoration of dune ecosystems.

Table 2. Determination of Auxin Production from PGPR

2-2. Gibberellin Productivity Coastal dunes PGPR Selection of

Among the sand dunes PGPR having excellent auxin-producing ability selected in Example 2-1, in addition to auxin, gibberellin producing strains having the greatest influence on germination of seeds were to be selected.

Since plant hormones auxin and gibberellin have different growth promoting effects on plants, strains that produce both plant hormones at the same time can be easily applied as PGPR for glomerular restoration, and the growth promoting effect of glomerular plants is also It was considered to be superior to the hormone producing strains. To investigate the production capacity of gibberellin, the UV spectrophotometric method described by Holdbrook et al. Was used [Lee et al., 1983]. The analytical method was performed as shown in FIG. 1, and GA ₃ (Sigma, USA) was used as a standard for quantification. The measurement results are shown in FIG. 2.

As a result of searching for gibberellin producing strains, it was determined that 14 strains among 23 strains of pre-selected high concentration auxin productivity multifunctional coastal glomeruli PGPR 23 had gibberellin production capacity (FIG. 2). In particular, strains P. aurantiaca IB5-5, IB5-8, IB5-10, and IB5-14 produced more than 5 mg / ml of gibberellin. These results are expected to be very valuable to apply to the estuary soils of coastal sand dunes if the growth promotion of sand dunes through the indoor port test is verified.

Example  3. Selected PGPR of Mycological  Properties and classification

Pseudomonas excellent in plant growth promoting hormone production and plant pathogenic fungi growth ability as a strain isolated from the root zone of sand dunes as in Examples 1 and 2 The aurantiaca IB5-10 were selected to deposit a P. aurantiaca IB5-10 (KACC 91561P) as of June 3, 2010 in South Korea Agriculture Microbial Resource Center. In the following experiment, P. aurantiaca IB5-10 was used to evaluate the environmental stress relaxation and plant growth promotion of sand dunes. The bacteriological properties of the deposited P. aurantiaca IB5-10 are shown in Table 3. In addition, for the classification of P. aurantiaca IB5-10, 16S rDNA was isolated and sequenced, and the results of 16S rDNA sequencing are shown in SEQ ID NO: 1.

[Table 3. Mycological Properties of P. aurantiaca IB5-10]

16S rDNA sequencing results of P. aurantiac a IB5-10 (SEQ ID NO: 1)

TGGTACCGTCCTCCCGAAGGTTAGACTAGCTACTTCTGGTGCAACCCACT

CCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGC

GACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTT

GCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTC

GCGGCTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCCCAG

GCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTG

TCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGG

ACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAG

CTGACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATC

CATCTCTGGAAAGTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGT

TGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAAT

TCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAAT

GCGTTAGCTGCGCCACTAAGAGCTCAAGGCTCCCAACGGCTAGTTGACAT

CGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGC

TTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGG

TGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCA

CCCTCTACCATACTCTAGCTCGCCAGTTTTGGATGCAGTTCCCAGGTTGA

GCCCGGGGATTTCACATCCAACTTAACGAACCACCTACGCGCGCTTTACG

CCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTG

GCACAGAGTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAATACTCACG

TATTAGGTAAGTACCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAG

ACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCC

AATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTC

CAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTG

AGCCATTACCTCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCG

CAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAG

CGTCCGTTTCCGGACGTTATCCCCCACTACCAGGCAGATTCCTAGGCATT

ACTCACCCGTCCGCCGCTCTCAAGAGAAGCAAGCTTCTCTCTACCGCTCG

ACTTGCATGTGTAGCCGCNCCC

Example 4 Production Capacity of Stress Relaxing Hormone ABA

ABA is a substance in which plants respond to environmental stresses such as drying or high salt, and is known as a plant hormone that induces resistance to plants by controlling pore opening and closing of plant leaves and invading pathogens. Thus, the present inventors measured the ABA production capacity produced by the selected strain.

First, the selected strains were purified and HPLC was used for this. The purification method for HPLC analysis is shown in FIG. 3. The organic solvent extract was used as a regulator, and the standard ABA was purchased from Sigma. In addition, for purification of ABA, after adjusting the culture supernatant of the glomerular root zone multifunctional PGPR strain, P. aurantiaca IB5-10, to pH 2.5 with 7 N HCl, ethyl acetate was adjusted to 1: 1 (v: v). After stirring for 15 minutes in a stirrer, the ethyl acetate layer and water layer containing ABA were separated. The ethyl acetate layer is a layer in which free ABA remains, washed three times with 1/2 volume of distilled water, and concentrated with a reduced pressure concentrator to perform HPLC analysis. The water layer was adjusted to pH 7.0 using 5 N NaOH, and then the same amount of ethyl acetate was added and stirred to recover the water layer. As shown in FIG. 3, the recovered water layer was adjusted stepwise again to pH 11.0, pH 7.0, and pH 2.0, and the same amount of ethyl acetate was added to recover the water layer, and the finally bound ABA was separated by concentration. It was. Finally, the concentrated sample was dissolved in 50% MeOH at a concentration of 1 μl / ml and used as a sample for HPLC. For HPLC, reverse-phase HPLC was used, a C ₁₈ column was used, the flow rate was 1 ml / min, and the solvent was eluted with a concentration gradient of 20% MeOH and 100% MeOH. Elution of the solvent in the concentration gradient was 20% MeOH for 0 to 5 minutes, 20 to 100% MeOH for 5 to 36 minutes, and 100% MeOH for 36 to 40 minutes. The measurement results are shown in Fig.

As shown in FIG. 4, as a result of HPLC analysis, it was confirmed that ABA was detected at a Retention time of 19 minutes. Therefore, if P. aurantiaca IB5-10, a coastal sand dune PGPR strain that produces ABA, is applied due to the nature of sand dunes under high temperature and dry conditions, water loss in plants and resistance to pathogens are reduced by controlling pore opening and closing of sand dunes. It is thought that the effect of improving the coastal glomerular plant growth can be obtained by inducing the activity.

Example  5. Environmental stress relief  hormone JA of Production capacity

JA is generally a plant hormone produced from higher plants, and is known as a derivative that induces resistance to plants in response to invasion of pathogens, mechanical injuries, and various environmental stresses. It is also involved in the defense system of plants caused by growth and microbial infection. Most of JA or Me-JA is produced by fungi except plants, and rarely is produced by bacteria that are PGPR. The production capacity of JA, one of these plant hormones, was measured in P. aurantiaca IB5-10.

In the extraction of JA, the culture supernatant of P. aurantiaca IB5-10 was used as a sample in the same manner as the extraction of ABA in Example 4. After adjusting the culture supernatant of P. aurantiaca IB5-10 to pH 4.5 with 7 N HCl, ethyl acetate was added at 1: 1 (v / v) and stirred in a stirrer for 15 minutes, followed by JA The ethyl acetate layer containing was separated. The separated ethyl acetate layer was washed three times with 1/2 volume of distilled water, and then concentrated under reduced pressure. The concentrated sample was dissolved in 50% MeOH at a concentration of 1 μl / ml and used as a sample for HPLC. HPLC analysis was performed using a C ₁₈ column by reverse-phase HPLC, flow rate was 1 ml / min, solvent was eluted with 0.1% trifluoroacetic acid (acefatrile) = 6: 4 (v / v). Go et al., 2006. Then, after GC-MS analysis into acetone (aceton) in the extracted sample to the extraction by using a stirrer for 20 minutes, and filtered on a filter was added to the _{^{[9, 10- 2 H 2]}} JA of 20 ng to the internal standard . 50 ml of distilled water was added thereto, and then concentrated under reduced pressure to evaporate all acetone, leaving only the aqueous layer. The concentrated residue was dissolved in 0.1 M potassium phosphate (pH 7.5), adjusted to pH 2.5 using 6 N HCl, and then shaken with a stirrer for 1 hour by adding 1 g of DEAE (Diethylaminoethyl) cellulose (cellulose). I was. The shaken filtrate was filtered, partitioned three times with chloroform, and then water was removed with Na ₂ SO ₄ and concentrated under reduced pressure. The concentrated residue was dissolved in a small amount of diethyl ether, and then passed through NH ₂ Cartridges, followed by chloroform: isopropanol (2: 1, v / v) and di Ethyl ether (Diethyl ether): eluted in the order of acetic acid (98: 2, v / v). The filtrate was concentrated under reduced pressure, dissolved in diethyl ether, transferred to 1 ml of reaction vial, dried with nitrogen gas at 40 ° C., and 60 μl Ethereal in a vial twice for 30 minutes. Methyl ester was induced by adding diazomethane and dried with nitrogen gas. The sample in the reaction vial was dissolved in 40 μl of anhydrous dichloromethane, and 1 μl of the sample was injected into GC-MS. For quantitative analysis of JA, m / z 83, 151, and 153 were identified as ions, and then quantified by the ratio of peak areas. The measurement results are shown in FIG. 5 (HPLC) and Table 4 (GC-MS).

As a result of HPLC analysis, JA production capacity of P. aurantiaca IB5-10, a glomerular PGPR, was identified as a clear single peak of 3 minutes retention time. In addition, as shown in Table 4, GC-MS results confirmed that 0.083 ng / ml of JA was produced in P. aurantiaca IB5-10. This is not only meaningful for the isolation and purification of JA-producing bacteria, but also for the environmental and environmental impacts of sand dunes when exposed to the dune soils of P. aurantiaca IB5-10, the selected coastal dune PGPR strain. It seems to contribute greatly to the resistance to mechanical and pathogenic stress.

Table 4. GC-MS Analysis of JA Production Capacity in P. aurantiaca IB5-10]

Example  6. Stress Relieving Substance Levan of Production capacity

Levan is a kind of fructan. Levan from microorganism regulates seed germination and storage capacity in plants and alleviates drought, low temperature and salt stress. It also plays a role in the storage of major nitrogen sources in plants. Due to the nature of coastal sand dunes, it is an absolutely necessary material for sand dunes that are exposed to large amounts of water evaporation due to solar heat, extreme temperature difference between day and night and malnutrition. In this regard, the present inventors measured the production ability of levan, which is a substance necessary for growth and stress relief of sand dunes.

First, P. aurantiaca IB5-10 was inoculated with YP broth (1% bacto-yeast extract, 1% bacto-peptone) to which 2% sucrose was added, followed by incubation at 30 ° C for 24 hours. After the culture was centrifuged for 20 minutes at 8000 rpm, the culture supernatant was recovered. In order to confirm the production of Levan, TLC and HPLC analysis were performed. TLC was added to the culture supernatant in silica gel 60 F254 plate 0.1 μl each, and then developed twice in butanol / acetic acid / water (5: 4: 1, v / v / v) developing solvent. After development, it was dried in a fume hood and then soaked in 95% methanol, 5% sulfuric acid, 0.3% N- (1-naphthyl) -ethylenediamine solvent, and heated at 120 ° C. for 10 minutes [Kim et al., 2003]. HPLC was used as a reverse-phase HPLC gel filtration column (Shodex Ionpack KS-802, 300 × 8 mm, Japan), flow rate 0.4 ml / min, the solvent was eluted with a third distilled water. Standards were levan, sucrose, sucrose, fructose and glucose, all purchased from Sigma. Measurement results are shown in FIGS. 6 to 7 and Table 5.

As shown in Figure 6, the multi-functional coastal dune PGPR strains of P. aurantiaca IB5-10 was started to produce levan by using sucrose (sucrose) after incubation for 12 hours, the control E. In case of coli , sucrose was not used. In addition, as a result of measuring levan production in P. aurantiaca IB5-10 for 12 hours and 24 hours incubation through HPLC analysis, as shown in FIG. 7 and Table 5, when P. aurantiaca IB5-10 was incubated for 12 hours, 6.60 The levan of mg / ml was produced, 7.65 mg / ml was produced in 24 hours culture. As a result of this, P. aurantiaca IB5-10 with levan production ability was applied to coastal sand dunes, which are located in a coastal sand dune where there is rapid temperature difference and water and nutrient deficiency. It is expected to be very effective to restore sand dunes by supplying them.

[Table 5. Levan Production in P. aurantiaca IB5-10]

Example  7. Environmental stress  Mitigation effect verification

We examined whether P. aurantiaca IB5-10 induces dry stress resistance of sand dunes in sand dunes that are always exposed to dry environmental stresses.

Through the indoor pot test, the growth status of the seaweed flower was investigated after treatment with P. aurantiaca IB5-10 in an artificially constructed dry stress environment without moisture supply. First, the experiments were divided into P. aurantiaca IB5-10 treatment and no treatment, and experiments were conducted in a constant temperature and humidity chamber at 5,000 lux, 30 ° C., 50%, and 12 hours for 30 days. After the water supply was performed so that it did not dry, the water supply was then stopped. P. aurantiaca IB5-10 was incubated at 30 ° C. at 200 rpm for 24 hours, followed by centrifugation of the culture solution to recover only the cells and diluted with sterile water once. The inoculation concentration of the cells was set to 10 ⁷ CFU / ml, and each experimental group was examined by statistical treatment after 20 ports, three repeated experiments. The observation results are shown in FIGS. 8 to 10 and Table 6.

As shown in FIG. 8 and Table 6, in the case of the untreated germ flower after 30 days under the dry stress, 50% of yellow flowers decreased, and the growth of the sour flower treated with P. aurantiaca IB5-10 Lasted. In addition, as shown in FIGS. 9 and 10, when 50 days had elapsed under dry stress, about 90% of the untreated germ flowers were dried, but the survival rate of the sour flower treated with P. aurantiaca IB5-10 was decreased. 50%.

[Table 6. Induction effect of dry stress resistance on tidal flowers (30 days)]

In addition, the resistance induction effect was observed using the barley to verify the drying and salt stress resistance induction effect in various coastal sand plants. Experimental conditions were carried out in the same manner as the sumflower, and 5% aqueous NaCl solution was used during water supply to add salt stress. The observation results are shown in FIGS. 11 to 12.

As shown in Figs. 11 and 12, in the case of the barley barley without treatment with the cells, 100% dried in 10 days after stopping 5% salt treatment and water supply, but the barley barley treated with P. aurantiaca IB5-10 Was found to represent 80% survival rate. These results show that P. aurantiaca IB5-10 is resistant to environmental stress in sand dunes which are always exposed to dry and high salt conditions. That is, by having resistance to sand dunes, the survival rate of sand dunes in the dry and high salt conditions may be improved, which may have a great effect in restoring the ecological ecosystem of the destroyed coastal dunes.

Example  8. For mass culture Commercial badge  Development and Optimal Microbial Culture Conditions

In order to formulate P. aurantiaca IB5-10 with the highest growth efficiency and dry stress resistance among multifunctional PGPR, carbon and nitrogen sources for mass culture were investigated.

Davis minimal broth (K ₂ HPO ₄ 0.7%, KH ₂ PO ₄ 0.2%, (NH ₄ ) ₂ SO ₄ 0.1%, Glucose 0.1%, Sodium citrate 0.05%, MgSO ₄ ㆍ 7H ₂ O 0.01%) Amylose, maltose, galactose, sucrose, dextrose, arabinose, molasses and corn starch as carbon sources In case of nitrogen source, ammonium sulfate, peptone, KNO ₃ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , soybean cake, corn steep liquor It was investigated using. The measurement results are shown in FIGS. 13 and 14.

As a result of the measurement, molasses was 10 ¹² CFU / ml for carbon source and ammonium sulfate was 10 ¹¹ CFU / ml for nitrogen source, and the mass culture medium was based on carbon source and nitrogen source. It was configured as shown in Table 7.

[Table 7. Composition of commercial medium for mass culture]

In addition, using the 8L jar fermentor (KF-8L, KoBioTech Co., Korea) in the culture medium for the culture conditions such as the inflow amount and the stirring speed of the air during the bulk culture, as a result of the stirring speed at 200 rpm The highest cell density was obtained at 20 hours and the highest cell density at 2.5 NI / min for the air injection. Accordingly, optimum culture conditions for mass culture were determined as shown in Table 8 below.

[Table 8. Culture conditions for mass culture]

Example  9. For mass culture Commercial  Effect verification

For the efficiency of mass production of cells, LB broth and cell growth were compared and used for the culture medium and culture conditions in the existing laboratory. Comparative medium was used for LB broth, the culture was recovered every 2 hours while incubating at 30 ℃ using 8L jar fermentor (KF-8L, KoBioTech Co., Korea), and the cell growth was examined by diluting smear.

As a result of comparing the cell production amount of the medium cultured and established as described above with the LB medium which is most used as the microbial culture medium in the laboratory (FIG. 15), the LB medium shows the 10 ¹¹ CFU / ml maximum cell density. However, the commercial medium developed in the present invention showed a maximum cell density of about 10 ¹² CFU / ml. Therefore, it is expected that the commercial medium of the present invention can reduce the unit cost of the medium for mass production of the multifunctional PGPR strain for formulation, and increase the production of cells to produce a formulation having a high cell density.

Example  10. Highly stable formulation method

For powder formulation using the carrier of P. aurantiaca IB5-10 strain, a carrier having the highest stability to the strain used was selected. The powder phase formulation method using a carrier is one of the most widely used methods for formulating microorganisms, and it is a method for maintaining the water activity using inorganic carriers and increasing the stability of microorganisms attaching microorganisms to carrier pores. Carriers used were H1800A, H1800B, H1800C of Cerite Korea, Zeolite of Rexem, Ilite of Dongchang Illite, NaY, NZ, ND7, NaX and NaAs were supplied from the company and used.

For carrier selection, the culture solution and the carrier were mixed in a sterile box at a ratio of 1: 2 (v / v), and the mixed composition was treated at 45 ° C. for 1 hour, after diluting and smearing in LB agar medium, and the number of viable cells was examined. . The measurement result is shown in FIG.

As shown in FIG. 16, zeolite, illite, and H1800C were selected for use in P. aurantiaca IB5-10 powder formulation experiments. Zeolite, Ilite, and H1800C are information on the carrier selected from the initial cell weight, compared to other carriers. Zeolite is supplied from Crude Oil Co., Ltd. and is composed of AI ₂ O ₃ , SiO ₂ , Consists of Fe ₂ O ₃ , CaO, MgO, K ₂ O, Na ₂ O, SO ₃ , TiO ₂ , LOI. Ilite is supplied from Dongchang Illite Co., Ltd. and has a thickness of 30 Å, 0.1 to 0.3 μm in diameter, 10 of ground spacing, specific gravity of 2.6 g / ml, and chemical composition of SiO ₂ , AI ₂ O _{3 ,} Fe _{2 O 3, CaO, MgO,} K 2 O, Na 2 O, P 2 O 5, TiO 2, is composed of LOI. H1800C is supplied from Celite Korea, and its chemical composition is composed of SiO _{2 ,} Al ₂ O _{3 ,} Na ₂ O, CaO, Fe ₂ O _3, K ₂ O, TiO _2, MgO, pH 6.5 ~ 7.3, Specific gravity is 0.15 g / ml.

As a powder formulation method, the P. aurantiaca IB5-10 strain was incubated for 20 hours at 30 ° C., 200 rpm, 2.5 NI / min using an 8L jar fermontor, and then the culture solution was recovered to select a zeolite, which is a carrier selected. After mixing aseptically with Ilite and H1800C at 1: 2 (v / v), respectively, the package was firstly sealed in polyethylene resin. Formulation samples were prepared as shown in FIG. 17 for saddle stability experiments during storage at high and low temperatures of powder formulations.

Example  11. Powder  stability

In order to investigate the stability of microorganisms during the storage, distribution, and storage of P. aurantiaca IB5-10 liquid and powder products according to the change over time, high and low temperature stabilization experiments were conducted according to the R & D standards and methods as shown in Table 9 below. Was carried out. In the case of high temperature saddle stability test, each formulation sample was stored at 30 ° C. and carried out in 3 replicates for 18 weeks. In the case of low temperature stabilization experiment, the preparation samples were stored at 0 ° C. for 7 days, and 5 samples of one repetition were prepared and diluted to LB agar every week to check the number of viable cells. The measurement results are shown in FIGS. 18 to 19.

[Table 9. Standardization Method for Changes over Time (Communication of Pesticides and Biochemical Pesticides)]

As a result of the measurement, it was possible to track the change over time in the treatment at 30 ℃ for powder formulation. In control and illite, the cell density began to halve at 10 weeks and showed a cell density of 10 ¹ CFU / ml at 18 weeks, whereas for zeolite, the cell density was 10 ⁹ CFU / ml at 18 weeks. Was maintained, the cell density of about 10 ⁶ CFU / ml even in the case of H1800C (Fig. 18). In addition, in the low temperature fermentation experiment at 0 ℃, in the control and illite treatment, it was confirmed that the cell density of about 10 times reduced after 7 days storage, P. aurantiaca using zeolite and H1800C In the case of IB5-10, it was confirmed that the cell density continued steadily without decreasing (Fig. 19). According to the test standard of change over time according to the Rural Development Administration, it is possible to set the drug warranty period to 1 year because the test standard at 30 ° C is recognized if the change over time at high temperature is not possible due to the characteristics of the raw materials. Therefore, zeolite was selected as the best carrier for the formulation of powders for the use of microbial agents for promoting the growth of sand dunes, and the stability of the following microorganisms was used. Microbial agents were used.

Example 12. Validation of the effect of microbial agents for promoting growth of sand dunes

In order to verify the effect of the preparation prepared using P. aurantiaca IB5-10, growth promoting experiments were performed in a constant temperature and humidity chamber at 30 ° C. and 50% humidity. Treatment concentration of the formulation was 10 ⁷ CFU / pot, and sand dunes used for growth promotion and salt stress resistance induction test were seeded from the coastal sand dunes of Hwajin Beach, East Coast. Swimming and cramp barley were used. The sand used in the pot was collected from the sand dunes gathering area of the East Coast Whale Fire Beach in order to make the same sand environment as the salt and nutrients of the dunes. 400 g of sand per pot was evenly dispensed, and after dispensing, the cold treated seeds were planted to a depth of 1 cm and germinated in a thermo-hygrostat at 30 ° C. and 50% humidity. After germination, when the cotyledon was unfolded, only the cypress flower pot of the same height was selected and used for the growth promoting experiment. Each experiment was made up to 20 pots, and repeated experiments were repeated three times, and then statistically measured for the effect of the preparation. Measurement results are shown in FIGS. 20 to 22 and Tables 10 to 12.

As shown in Fig. 20 and Table 10, in the case of P. aurantiaca IB5-10 powder formulation, after 10 days in the cypress flowers, the stem elongation was 1.5 times, the root length 1.4 times, the number of roots 1.8 times, and the leaf of the main leaf compared to the untreated group. It was measured to be 2.2 times more. As shown in FIG. 21 and Table 11, in the P. aurantiaca IB5-10 powder formulation, the germination rate was 4.9 times higher and the leaf area was about 50% higher than in the untreated group. As shown in FIG. 22 and Table 12, the P. aurantiaca IB5-10 powder formulation in the barley barley test was investigated to have 2 times the height of the stem, 5.2 times the root length, 2.1 times the number of roots, and 2.2 times the length of the main leaf. It became. Therefore, the P. aurantiaca IB5-10 powder formulation developed in the present invention has been proved to have an excellent effect on the growth of coastal sand dune plants.

[Table 10. Growth Promotion Effect on the Seaweed Flower]

[Table 11. Growth Promotion Effects on the Swordfish Swimming]

Table 12. Growth Promotion Effects on Barley

Example  13. A method for restoring sand dunes using microbial agents for promoting growth of sand dunes

In order to verify the possibility of sand dunes restoration in actual coastal sand dunes of preparations made using multifunctional PGPR, a practical application test was performed. Field application test was carried out in Gyeongsangbuk Pohang Hwajin beach 50 four coast in the training four that are accessible to the public limited (Fig. 23), the experiment 10 the gaetme flower seeds 60 ribs per treatment of the powder formulation and then implanted in the water ⁷ CFU / Dilution to ml was followed by spraying.

First, on July 29, 2009, a square ball was installed to sow 60 seedling flower seeds. The germination rate survey was then conducted on September 29, 2009, 60 days later. In the untreated group, the germination rate was 0%, while the P. aurantiaca IB5-10 formulation treated group was found to be 36.6% (FIGS. 24 to 25, Table 13). The germination of the P. aurantiaca IB5-10 formulation developed in the present invention during the non-natural germination period, and the germination rate close to 40% compared to the untreated group, is considered to have an excellent effect in actual coastal sand field. If it is fertilized during the proper germination period of coastal sand dunes, it is considered to be of great help in promoting coastal dune plant growth and germination for restoration of coastal sand dunes destroyed due to the improvement of germination rate and growth of dune plants. It is expected that the microbial preparation using multifunctional PGPR not only has an excellent effect but also has a great effect in reducing coastal restoration cost and restoring eco-friendly ecosystem in the future.

Table 13. Results of Germination Flower Germination by Microbial Agents for Promotion of Sand Dune Plant Growth]

Agricultural Biotechnology Research Institute KACC91561P 20100603

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Claims (12)

delete delete delete KACC 91561P isolated from the rhizosphere of sand dunes and having the production capacity of abscisic acid and jasmonic acid as environmental stress mitigating hormones and levan as environmental stress mitigating agent. Microbial agent for environmental stress relief containing strain Pseudomonas aurantiaca IB5-10 deposited as an active ingredient.
delete The method of claim 4, wherein the microbial agent is a microbial agent for reducing environmental stress, characterized in that the culture solution of Pseudomonas aurantiaca IB5-10 is mixed with an inorganic carrier.
According to claim 6, wherein the culture medium Pseudomonas aurantiaca ( Pseudomonas aurantiaca ) IB5-10 molasses (Molasses) 15-30 g / L, Corn steep liquor (15-30 g / L), ammonium sulfate ( Ammonium sulfate 5-15 g / L, Monopotassium phosphate 4-7 g / L, Dipotassium phosphate 3-5 g / L, Magnesium sulfate 2-4 g / L Manganese sulfate (Manganese sulfate) 0.3-0.7 g / L, antifoaming agent (Antiforming agent) microbial agent for reducing environmental stress, characterized in that cultured in a medium containing 0.5-0.8 g / L.
The zeolite of claim 6, wherein the inorganic carrier comprises AI ₂ O ₃ , SiO ₂ , Fe ₂ O ₃ , CaO, MgO, K ₂ O, Na ₂ O, SO ₃ , TiO ₂ , and LOI. Ilite consisting of SiO 2, AI ₂ O _{3 ,} Fe ₂ O ₃ , CaO, MgO, K ₂ O, Na ₂ O, P ₂ O ₅ , TiO ₂ , and LOI, or SiO _{2 ,} Al ₂ O _{3 ,} Na ₂ O, CaO, Fe ₂ O _{3 ,} K ₂ O, TiO _{2 ,} and Microbial agent, characterized in that H1800C consisting of MgO.
A method for increasing environmental stress resistance of sand dunes comprising administering to the sand dunes the microbial agent for environmental stress relief according to claim 4.
10. The method according to claim 9, wherein the administration of the glomerular plant is carried out by diluting the microbial agent with water and then spraying it.
delete 10. The method according to claim 9, wherein the dune plant is a magnolia flower, a magpie swimming or a barley barley.

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