KR102551903B1 - Novel Pseudomonas guariconensis ECO-K-3-3 having potassium solubilizing activity and its optimal activity culture condition - Google Patents

Abstract

The present invention relates to a novel Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) having potassium solubilizing activity and its optimal activity culture conditions, Pseudomonas guariconensis selected in the present invention ( Pseudomonas guariconensis ) ECO-K-3-3 (KACC 92484P) strain has high solubilizing potassium activity. In addition, it exhibits excellent potassium solubilizing activity under the cultivation conditions of 1% peptone, 1% maltodextrin, and 0.3% sodium chloride, thereby exhibiting the effect of preventing salt accumulation in the soil of facility cultivation and being economically efficient. Optimal culture conditions can be established.

Description

Novel Pseudomonas guariconensis ECO-K-3-3 having potassium solubilizing activity and its optimal activity culture condition

The present invention relates to a novel Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) having potassium solubilizing activity and its optimal active culture conditions, and more particularly to a novel Pseudomonas guariconensis having potassium solubilizing activity It relates to Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3 and several culture conditions showing its potassium solubilizing optimum activity.

As people's living standards and income increase, interest in health has increased, and the ownership of high-quality fresh vegetables has been enjoyed, and the area of facility vegetable cultivation that can be grown throughout the year is increasing. For example, domestic facility cultivation increased by more than 20 times from 4,000 ha in the 1970s to 83,000 ha in 2002 (Reference: Oh Sang-eun, Son Jeong-soo, Ok Yong-sik, Joo Jin-ho, Plans to reduce salt accumulation by soil desalination and drainage in facility cultivation. Journal of the Korean Soil Fertilizer Society, 2010, 43, 443-49).

Due to the intensive cultivation characteristics of the facility house, there is a problem that the use of chemical fertilizers and compost increases, and the excessive fertilized fertilizer components accumulate in the soil to increase the salt concentration, resulting in salt accumulation. When salts accumulate, the osmotic pressure of the soil solution increases and the difference in osmotic pressure between the roots of the crop decreases. As a result, water absorption becomes poor, and the amount of water transpiration from the leaves cannot be absorbed and supplied by the roots, resulting in a lack of moisture in the crop. In severe cases, moisture from the crop body may be deprived (References: Hong Seong-gu, Lee Nam-ho, Jeon Woo-jung, Hwang Han-cheol, Kim Jin-tae, Salt accumulation phenomenon and decontamination method in facility cultivation soil. Abstracts of Korean Society of Agricultural Engineering Conference, 1999, 447-53).

So far, as a method to solve the salt accumulation phenomenon in the facility cultivation area, there are irrigation decontamination, subsoil inversion method, subsoil fracturing, sucking crops, and topsoil removal reduction method, but these methods have various problems such as short-term effects, uneconomical maintenance, and groundwater contamination there is In addition, even in the case of microbial preparations known to date, their effectiveness is extremely negligible during on-site action (Reference: Oh Sang-eun, Son Jeong-su, Joo Jin-ho, Methods to reduce salt accumulation by soil desalination and drainage in facility cultivation. Journal of the Korean Soil Fertilizer Society, 2010 , 43, 443-49).

Microbial preparations are harmless to soil plants and humans, and can be used regardless of the growing season because there is no residual problem or drug damage, and there is no tolerance and crop stress. Therefore, countermeasures against salt accumulation in facility cultivation are urgently needed, and it is necessary to research and develop new and excellent microbial agents to solve this problem.

Republic of Korea Patent Registration No. 10-1281104 (registration date: 2011.03.25) relates to a microbial preparation containing useful soil microorganisms and a method for restoring salinity-damaged soil using the same, belonging to the genus Pantoea deposited under accession number KCTC 11893BP J11 (Pantoea sp. J11) strain, a composition for relieving soil salt accumulation containing the same, a plant growth promoter containing the same, a method for relieving salt accumulation comprising treating the soil with a composition containing the same, and a composition containing the same for plants Or a plant growth promotion method comprising the step of treating the soil in which the plant is grown is described.

The present invention is to provide a novel Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) with excellent potassium solubilizing activity and its optimal active culture conditions in order to solve the salt accumulation of facility cultivation.

The present invention provides a Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) strain or a lyophilized product of the strain, which exhibits potassium solubilizing activity.

In addition, the present invention provides a culture solution of Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) strain or a lyophilized product of this strain culture solution, which exhibits potassium solubilizing activity.

In addition, the present invention is inoculated with Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) in a medium containing peptone, maltodextrin, and sodium chloride. It provides a culture method characterized by culturing.

In addition, the present invention Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3 (KACC 92484P) strain or a culture of this strain or a lyophilisate of this strain or a lyophilisate of this strain culture, containing potassium Provided is a soil salt accumulation prevention method characterized by spraying the salt accumulation soil.

The Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) strain selected in the present invention has high solubilizing potassium activity. In addition, it exhibits excellent potassium solubilizing activity under culture conditions containing peptone, maltodextrin, and sodium chloride, thereby exhibiting the effect of preventing salt accumulation in the soil of greenhouse cultivation, while being economically efficient and optimal culture conditions can establish.

Figure 1 is a photograph of the result of confirming the potassium solubilization activity of the strains isolated from the culture medium having a color change in the staining medium.
FIG. 2 is a photograph of a medium obtained by measuring the potassium solubilizing activity of the strains having the potassium solubilizing activity selected in FIG. 1 by a paper disc assay.
Figure 3 is a graph (A) and a medium photograph (B) of the results of measuring the pH-specific activity of Pseudomonas guariconensis ECO-K-3-3 according to the present invention using a paper disk assay.
4 is a graph (A) and a photograph (B) of the medium as a result of measuring the temperature-specific activity of Pseudomonas guariconensis ECO-K-3-3 of the present invention by a paper disk assay.
Figure 5 is a graph (A) and a photograph (B) of the results of measuring the activity over time of the present invention Pseudomonas guariconensis ECO-K-3-3 using a paper disk assay.
Figure 6 is a graph (A) and a photograph (B) of the results of measuring the activity of each type of carbon source of Pseudomonas guariconensis ECO-K-3-3 of the present invention by a paper disk assay.
Figure 7 is a graph (A) and a photograph (B) of the results of measuring the activity of Pseudomonas guaiconensis ECO-K-3-3 according to the concentration of maltodextrin according to the present invention using a paper disk assay.
Figure 8 is a graph (A) and a photograph (B) of the results of measuring the activity of Pseudomonas guaiconensis ECO-K-3-3 according to the nitrogen source type and concentration of the present invention by a paper disk assay.
Figure 9 is a graph (A) and a photograph (B) of the results of measuring the activity of Pseudomonas guariconensis ECO-K-3-3 of the present invention by sodium chloride (NaCl) concentration by paper disk assay.

Due to the intensive cultivation characteristics of the facility house, there is a problem that the use of chemical fertilizers and compost increases, and the excessive fertilized fertilizer components accumulate in the soil to increase the salt concentration, resulting in salt accumulation. When salts accumulate, the osmotic pressure of the soil solution increases and the osmotic pressure difference between the roots of crops decreases, resulting in poor water absorption. In severe cases, there is a problem that moisture in the crop body is deodorized. Existing methods for solving salt accumulation have had several limitations. Among them, the use of microbial preparations is harmless to soil plants and humans, and can be used regardless of the growing season because there is no residual problem or drug damage, and tolerance and crop stress There is an advantage that there is no, but at present, there are very few effective preparations, so new research and development is required for using new microbial preparations.

Accordingly, the present invention provides a Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) strain or a lyophilized product of the strain, which exhibits a potassium solubilizing activity. In addition, the present invention provides a culture solution of Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) strain or a lyophilized product of this strain culture solution, which exhibits potassium solubilizing activity. To this end, in the present invention, strains exhibiting excellent potassium solubilizing activity were isolated from strains collected by taking samples from farm soil near the Jeongeup Agricultural and Livestock Microbial Industry Development Support Center, and Pseudomonas guariconensis was analyzed through 16S rRNA sequencing. ( Pseudomonas guariconensis ), and it was named Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3 (KACC 92484P).

At this time, in the present invention, 'freeze drying' is not limited in any way as long as it is known in the art, and since it corresponds to a conventional technique, a detailed description thereof will be omitted.

In addition, the present invention is inoculated with Pseudomonas guariconensis ECO-K-3-3 (KACC 92484P) in a medium containing peptone, maltodextrin, and sodium chloride. It provides a culture method characterized by culturing. At this time, in the medium, it is preferably composed of 0.5 to 1.5% of the peptone, 0.5 to 1.5% of maltodextrin, and 0.3 to 0.5% of sodium chloride, more preferably 1% of the peptone and 1% of maltodextrin. , preferably composed of 0.3% sodium chloride. Through this, the strain can exert a more excellent potassium solubilizing activity. This composition of the medium was found through experiments as a result of diligent efforts to find the conditions for optimal activity for each carbon source, nitrogen source type and concentration, and sodium chloride concentration, and through this, peptone (0.5%), beef extract (0.3%), and sodium chloride were found. (0.5%) in the existing Aleksandrov medium (References: Rajawat MVS, Singh S, Tyagi SP, Saxena AK. A modified plate assay for rapid screening of potassium-solubilizing bacteria. Pedosphere, 2016, 26, 768-73). In comparison, potassium solubilizing activity is excellent (13% increase), and cost is saved (40% decrease), so it is possible to provide an excellent medium in terms of economy.

On the other hand, when the Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3 (KACC 92484P) is cultured, it is preferably cultured at pH 5-7, 30-40 ° C, 48-96 hours, more preferably It is preferable to incubate at pH 6, 35 ℃, 72 hours. Through this, the strain can exert a more excellent potassium solubilizing activity.

In addition, the present invention Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3 (KACC 92484P) strain or a culture of this strain or a lyophilisate of this strain or a lyophilisate of this strain culture, containing potassium Provided is a soil salt accumulation prevention method characterized by spraying the salt accumulation soil. In this way, by directly spraying potassium-containing salts on the soil, insoluble or mineral-structured potassium compounds are removed. As a soil solution, it is converted into a soluble form in the soil to be used by plants (Ref. X Zeng, X Liu, J Tang, S Hu, P Jiang, W Li. Characterization and potassium-solubilizing ability of Bacillus circulans Z1-3 Adv Sci Lett, 2012, 173-76), can solubilize calcium in calcium-minerals and improve plant growth and yield (Ali AM, Awad MY, Hegab SA, Gawad AMAE, Eissa, MA. Effect of potassium solubilizing bacteria (Bacillus cereus) on growth and yield of potato.Journal of Plant Nutrition, 2021, 411-20).

On the other hand, according to the following experiments, Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3 selected in the present invention has excellent potassium solubilization activity, and according to finding the optimal activity conditions for each pH, temperature and time It was possible to establish the culture conditions showing the most excellent potassium solubilizing activity. In addition, by finding the optimal activity conditions for each carbon source type and concentration, nitrogen source type and concentration, and sodium chloride concentration, it was possible to establish the culture conditions showing the best potassium solubilization activity. Through this, by establishing the composition of the medium showing the optimal activity, it was possible to obtain an excellent medium in terms of economy while having excellent potassium solubilization activity compared to the existing medium, which can be applied in a variety of ways, such as when cultivating facilities in the agricultural industry.

Hereinafter, the contents of the present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

[Example 1: Novel Pseudomonas guariconensis with excellent potassium solubilization activity ( Pseudomonas guariconensis ) Isolation and identification of ECO-K-3-3]

In this example, a novel strain having excellent potassium solubilizing activity was isolated and identified.

1) Novel Pseudomonas guariconensis ( Pseudomonas guariconensis ) Isolation and identification of ECO-K-3-3

Samples were taken from farm soil near the Jeongeup Agricultural and Livestock Microbial Industry Development Support Center, and soil samples were added to Aleksandrov-modified dyeing medium (see Table 1) and cultured at 35 ° C. for more than 3 days. Assuming that there is a potassium-solubilizing active strain in the cultures with color changes in the staining medium, a total of 20 strains were isolated by single colony isolation of the cultures, and potassium solubilization of the isolated strains (first screening) activity was investigated. The results were the same as in FIG. 1 .

Accordingly, strains having potassium solubilizing activity selected above were cultured in Aleksandrov medium at 30° C. for 24 hours, and potassium solubilizing activity was measured in the following manner: First, a paper disc was placed on Aleksandrov modified staining medium. After centrifugation of the culture solution of the strains having potassium solubilizing activity selected above, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. Potassium solubilizing activity was determined by observing the zone size. The detailed composition of the Aleksandrov medium and the Aleksandrov modified staining medium was shown in Table 1, and the same medium was used below.

Aleksandrov medium (pH7.0) Aleksandrov modified staining medium (pH7.2) Peptone 0.5% Glucose 0.5% Beef extract 0.3% Magnesium sulphate 0.05% Sodium chloride 0.5% Ferric chloride 0.0005% Calcium carbonate 0.01% Calcium phosphate 0.2% K-bearing minerals 0.2% Bromocresol purple 0.3% Phenol red 0.3% Bromothymol blue 0.3%

As a result, as shown in FIG. 2, the strains exhibiting high potassium solubilization activity at 2.9cm and 2.6cm were confirmed to be pathogenic strains, but had high potassium solubilization activity, but were found not to be suitable for selection as the final strain. Accordingly, it was found that the strains exhibiting the highest potassium activity were strains (non-pathogenic) with potassium activity of 2 cm. As a result of confirming the non-pathogenic strains (potassium activity of 2 cm), all of them were the same strain, and among them, 'ECO -K-3-3' was selected as the final strain. Accordingly, identification was performed through 16S rRNA sequencing for the finally selected strain 'ECO-K-3-3'. After pure isolation, the isolated strain was requested to Microgen (Daejeon) using refrigerated delivery in a plate state, and 16S rRNA sequencing was performed using universal primer sets of 27F and 1492R. . A total 1,261 bp base sequence (SEQ ID NO: 1) of the 16S rRNA coding base sequence of the isolate 'ECO-K-3-3' was determined. Based on the base sequence, the homology test of the base sequence was performed by the Blast program (http://www.ncbi.nlm.nhiv.gov) for the registered information (GeneBank database). As a result, Pseudomonas guariconensis ( Pseudomonas guariconensis ) It was confirmed that the species exhibits homology that matches 99%.

Therefore, the inventors of the present invention, as of November 28, 2022, to the Microbial Bank (KACC) of the National Academy of Agricultural Sciences, Rural Development Administration of the Republic of Korea for the identified strain, Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3 (Accession No. : KACC 92484P).

[Example 2: Pseudomonas gua conensis of the present invention ( Pseudomonas guariconensis ) Establishment of optimal culture conditions for ECO-K-3-3]

In this example, an experiment was conducted to establish optimal culture conditions exhibiting high potassium-solubilizing activity of the selected Pseudomonas guariconensis ( Pseudomonas guariconensis ) ECO-K-3-3.

1) Activity by pH

Aleksandrov medium was adjusted to pH 4-9, respectively, and Pseudomonas guaiconensis ECO-K-3-3 cultured at 30 ° C for 24 hours was measured for potassium solubilizing activity in the following manner: First, on Aleksandrov modified staining medium A paper disk was placed. After centrifuging the culture solution of the cultured strain, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. The area size was observed to determine potassium solubilizing activity. As a result, as shown in FIG. 3, it was confirmed that the highest potassium activity appeared at 2.0 cm under pH 6 conditions.

2) Activity by temperature

Pseudomonas guaiconensis ECO-K-3-3 cultured in Aleksandrov medium at 25 ° C, 30 ° C, 35 ° C, and 40 ° C for 24 hours, respectively, was measured for potassium solubilizing activity in the following way: First, Aleksandrov modified staining medium A paper disk was placed on top. After centrifuging the culture solution of the cultured strain, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. The area size was observed to determine potassium solubilizing activity. As a result, as shown in FIG. 4, it was confirmed that the highest potassium activity was observed at 3.0 cm at 35 ° C.

3) Active by hour

Potassium solubilizing activity of Pseudomonas guaiconensis ECO-K-3-3 cultured in Aleksandrov medium at 35°C for 24 hours, 48 hours, 72 hours, and 96 hours, respectively, was measured in the following manner: First, Aleksandrov modified staining medium A paper disk was placed on top. After centrifuging the culture solution of the cultured strain, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. The area size was observed to determine potassium solubilizing activity. As a result, as shown in FIG. 5, it was confirmed that the highest potassium activity appeared at 3.2 cm under 72-hour conditions.

4) Activity by type of carbon source

Pseudomonas guariconensis ECO-K-3 cultured at 35°C for 72 hours by adding 1% glucose, 1% sucrose, and 1% maltodextrin to 1% nitrogen source peptone, respectively. Potassium solubilizing activity of -3 was measured in the following manner: First, a paper disk was placed on Aleksandrov's modified staining medium. After centrifuging the culture solution of the cultured strain, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. The area size was observed to determine potassium solubilizing activity. As a result, as shown in FIG. 6, it was confirmed that the highest potassium activity was observed at 2.8 cm under the condition of 1% maltodextrin.

5) Activity by maltodextrin concentration

Potassium solubilization activity of Pseudomonas guaiconensis ECO-K-3-3, which was cultured at 35°C for 72 hours by adding 0.5 to 2.5% of maltodextrin to 1% of nitrogen source peptone, respectively, was measured in the following manner: First Paper discs were placed on top of Aleksandrov's modified staining medium. After centrifuging the culture solution of the cultured strain, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. The area size was observed to determine potassium solubilizing activity. As a result, as shown in FIG. 7, the highest potassium activity was shown at 1.6 cm in the conditions of 1% maltodextrin and 1.5% maltodextrin, and the condition of 1% maltodextrin was determined as the optimal condition in consideration of economical efficiency.

6) Activity by nitrogen source type and concentration

Pseudomonas guariconensis ECO-K-3-3 cultured at 35℃ for 72 hours by adding 0.5~1.5% of nitrogen source peptone and 0.5~1.5% of yeast extract to 1% of carbon source maltodextrin, respectively, was prepared in the following manner. Potassium solubilizing activity was measured: Paper disks were first placed on Aleksandrov's modified staining medium. After centrifuging the culture solution of the cultured strain, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. The area size was observed to determine potassium solubilizing activity. As a result, as shown in FIG. 8, it was confirmed that the highest potassium activity was observed at 1.8 cm in the peptone 1% condition.

7) Activity by sodium chloride (NaCl) concentration

Potassium solubilizing activity of Pseudomonas guaiconensis ECO-K-3-3, which was cultured at 35°C for 72 hours by adding 0.1 to 0.5% sodium chloride to 1% carbon source maltodextrin, 1% nitrogen source peptone, respectively, was measured by the following method. : First, a paper disc was placed on the Aleksandrov modified staining medium. After centrifuging the culture solution of the cultured strain, 40 μl of the supernatant was dispensed onto paper disks, followed by static culture at 35° C. for 72 hours. The area size was observed to determine potassium solubilizing activity. As a result, as shown in FIG. 9, it was confirmed that the highest potassium activity appeared at 1.8 cm in the condition of 0.3% sodium chloride.

[Experimental Example 1: Aleksandrov medium and Pseudomonas guariconensis of the present invention ( Pseudomonas guariconensis ) Efficiency comparison of optimal culture medium conditions of ECO-K-3-3]

In this experimental example, the efficiency of the Aleksandrov medium and the optimal culture medium conditions established above for Pseudomonas guariconensis ECO-K-3-3 of the present invention were compared. As a result, as shown in Table 2, compared to Aleksandrov medium, the optimal culture medium conditions of the present invention increase potassium solubilization activity by 13% (1.6 cm → 1.8 cm), and are economical in terms of price (793.8 won / L → 319.2 won / L) was confirmed, and through this, it was found that optimal culture medium conditions having excellent potassium solubilization activity were established.

Aleksandrov medium (pH7.0) Optimal medium (pH6.0) condition Peptone 0.5% condition Peptone 1% Beef extract 0.3% Maltodextrin 1% Sodium chloride 0.5% Sodium chloride 0.3% Potassium
solubilizing activity 1.6cm Potassium
solubilizing activity 1.8cm price 793.8 Won/L price 319.2 Won/L

Rural Development Administration National Academy of Agricultural Sciences Microorganism Bank (KACC) KACC92484P 20221128

Claims (4)

Pseudomonas guariconensis exhibiting potassium solubilizing activity, cultured in a medium containing 0.5 to 1.5 wt% of peptone, 0.5 to 1.5 wt% of maltodextrin, and 0.3 to 0.4 wt% of sodium chloride ( Pseudomonas guariconensis ) Culture broth of ECO-K-3-3 (KACC 92484P) strain.
Pseudomonas guariconensis exhibiting potassium solubilizing activity, cultured in a medium containing 0.5 to 1.5 wt% of peptone, 0.5 to 1.5 wt% of maltodextrin, and 0.3 to 0.4 wt% of sodium chloride ( Pseudomonas guariconensis ) ECO-K-3-3 (KACC 92484P) A composition for relieving soil salt accumulation comprising a culture solution of the strain or a freeze-dried product thereof.
In a medium containing 0.5 to 1.5% by weight of peptone, 0.5 to 1.5% by weight of maltodextrin, and 0.3 to 0.4% by weight of sodium chloride, Pseudomonas guariconensis ECO-K- A culture method characterized by inoculating and culturing 3-3 (KACC 92484P).
Pseudomonas guariconensis cultured in a medium containing 0.5 to 1.5% by weight of peptone, 0.5 to 1.5% by weight of maltodextrin, and 0.3 to 0.4% by weight of sodium chloride Pseudomonas guariconensis ECO-K -3-3 (KACC 92484P) A method for preventing salt accumulation in soil, characterized in that by spraying a culture solution of the strain or a lyophilized product thereof to a salt-rich soil containing potassium.

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