KR20080058522A - Pseudomonas sp. cl-1 and method of solubilizing insoluble-phosphate by using the same - Google Patents

Abstract

A novel strain is provided to solubilize insoluble-phosphate excellently within a short time, prevent solubilized phosphate from being fixed on soil due to reusing the solubilized phosphate at a rapid rate, and supply phosphoric acid, which is necessary for a plant, efficiently by gradually releasing the solubilized phosphate into the soil again. A Pseudomonas sp. CL-1 strain solubilizing insoluble-phosphate fixed on soil is deposited as a deposition no. KACC 91282P. A biofertilizer comprises a fungus body or a culture solution of the Pseudomonas sp. CL-1 strain. To solubilize the insoluble-phosphate fixed on the soil, the strain or the fertilizer is directly applied to the soil.

Description

Novel Pseudomonas sp. CL-1 strain and solubilization method of poorly soluble phosphate using the same {Pseudomonas sp. CL-1 and Method of solubilizing insoluble-phosphate by using the same}

Figure 1 shows the solubilization capacity of tricalcium phosphate of Pseudomonas sp. CL-1 strain in a solid medium.

Figure 2 shows the identification results using the 16S rRNA gene sequence of Pseudomonas SP CL-1.

Figure 3 shows a phylogenetic analysis using the 16S rRNA gene sequence of Pseudomonas SP C-1.

Figure 4 shows a phase contrast micrograph of Pseudomonas SP CL-2.

The present invention provides a Pseudomonas SP C-1 excellent in solubilizing the poorly soluble phosphate fixed in the soil, and also relates to a method for solubilizing poorly soluble phosphate using the strain as an active ingredient.

Phosphoric acid is one of the three fertilizers, along with nitrogen, phosphoric acid and kali as essential elements for plants and is an essential ingredient for cell growth and reproduction. In the form of inorganic phosphate fertilizers, phosphate ore is manufactured as a main raw material, and Korea has imported all of it from abroad because phosphate is not produced (Lim, Characteristics and Effect for Application of Phosphate Rock as a Source of Phosphate Fertilizer , 1993, p 1).

In general, phosphate fertilizers are different from nitrogenous and caliber fertilizers, so they are not easily dissolved or lost when applied to soil. Even if phosphorus-containing inorganic phosphate fertilizer, which is readily available to plants, is applied to the soil, it is reported that the ratio of fixing to the soil is higher than that of plants. Most of the phosphorus used in the form of Ca-P, Fe-P, Al-P is fixed to the soil and bound with poorly soluble phosphate, which is difficult for plants to use. (National Institute of Agricultural Technology, Soil Chemical Analysis , 1988, p73). Particularly, the distribution of fractional phosphorus to total phosphorus in plant cultivated soils reported that Ca and Fe-bonded phosphates accounted for the majority of inorganic phosphates in the order of Ca-P>Fe-P>residual-P>Al-P> saloid-P. (Suh, Biological recycling of the insoluble phosphates accumulated in cultivated soils by the phosphate-solubilzing microorganisms , 1994, p98)

As such, the inorganic phosphate fertilizer in the form of plants that are readily available to the soil is applied to the soil, but the phosphate fertilizer is well fixed in the soil, so the phosphate fertilizer is continuously applied every time the crop is grown. As a result, the accumulated phosphate in the soil multi-cultivated throughout the year results in far exceeding the proper content, resulting in a soil environment that inhibits soil contamination and crop growth. In Korea, soil phosphate content in facility horticulture media is mostly accumulated above the proper content.

Considering the fact that phosphate fertilizer is produced and sold at a higher price than other fertilizers, there is an urgent need for a method of efficiently using a large amount of poorly soluble phosphate accumulated in farmland.

For this purpose, it is necessary to maximize the utilization by using the material circulation function of the soil microorganisms. Furthermore, there is a need for the selection of excellent microbial strains that can solubilize poorly soluble phosphate immobilized in the soil with good efficiency and settle well in the soil and plant root zones. Among known phosphate solubilizing bacteria to date Pseudomonas (Pseudomonas), Bacillus (Bacillus), Agrobacterium (Agrobacterium), ssanto Pseudomonas (Xanthomonas), Flavobacterium (Flavobacterium), Brevibacterium (Brevibacterium), Achromobacter (Achromobacter), O (Pantoea), bakteo (Aerobacter) in the Oh, micro Cocos (Micrococcus), ssera thiazole (Serratia), Enterobacter bakteo (Enterobacter), alkali jeneseu (Alcaligenes), ewiniah (Erwinia) in panto, Escherichia It is reported that the genus Escherichia has phosphate solubilizing ability (Subba-Rao, Advances). in Agricultural Microbiology : Phosphate Solubilization by Soil1 Microorganisms . Butterworth , London . 1982, p 293-303).

 However, the phosphate solubilization ability of the microorganisms introduced above varies considerably according to the type, and the strains belonging to the same genus also show a remarkable difference in phosphate solubility, depending on the species. Do.

Therefore, there is a need for strain selection having excellent functions to effectively solubilize the poorly soluble phosphate fixed in the soil, reduce the amount of phosphate fertilization and increase fertilization efficiency.

It is an object of the present invention to provide Pseudomonas sp. CL-1 strain, which is an excellent strain solubilizing poorly soluble phosphate immobilized in soil. In addition, the present invention provides a method for effectively solubilizing the poorly soluble phosphate fixed in the soil using the strain of the invention and biologically blocking the fixation to the soil by using the characteristics of the strain to reuse the solubilized phosphorus in the microbial composition killing the strain Effective use of soluble phosphorus, which is continuously released in the process.

It is an object of the present invention to provide Pseudomonas sp. CL-1 strain, which is an excellent strain solubilizing poorly soluble phosphate immobilized in soil. It is also to provide a method for effectively solubilizing poorly soluble phosphate fixed in the soil.

The strain of the present invention is a strain having a superior function of phosphate solubilizing ability in a short time, and biologically blocking the fixation to the soil by reusing solubilized phosphorus in the composition of the microorganism, and slowly released into the soil again in the process of killing the strain of the present invention The required phosphoric acid component is efficiently supplied.

It was identified as Pseudomonas sp. Strain by molecular lineage analysis, and deposited on the Korea Agricultural Microorganisms Preservation Center (KACC 91282P), an international depository institution, on November 14, 2006.

Hereinafter, the present invention will be described in detail with reference to Examples, but the protection scope of the present invention is not limited to the following Examples.

Example  1.: Isolation and Selection of Strains

The roots and root soils of plants were collected from Nonsan, Neum, Cheongju, and Chungju vegetable cultivation media (cucumbers, lettuce, tomatoes, peppers) to isolate bacteria with excellent phosphate solubilization. The collected sample was put in sterile saline solution and shaken at 240 rpm for 10 minutes, and the suspension was diluted stepwise at 10 ¹ to 10 ⁷ magnification. The dilution was taken from a solid medium containing soluble phosphate [calcium phosphate, Ca ₃ (PO ₄ ) ₂ ] Pickovskaya's medium: Ca ₃ (PO ₄ ) ₂ 5 g, glucose 10 g, (NH ₄ ) ₂ SO ₄ 0.5g, KCl 0.2g, MnSO ₄ 0.01g, Agar 15g, distilled water 1L). Strains solubilizing poorly soluble phosphate were purely isolated while incubated at 30 ° C. for 5 days. The pure strain was re-inoculated into the medium used above, and then the size of the transparent ring was selected to select the best strain to solubilize poorly soluble phosphate. The selected CL-1 strains were isolated from the red pepper roots.

Example  2.: Identification of the strain of the present invention

Identification of the strains selected in Example 1 was performed using morphological and biochemical characteristics and 16S rRNA gene sequencing.

(1) Morphological and biochemical characterization

 The strain isolated in Example 1 grows well in Tryptic Soy Agar medium and the morphology of bacteria was slightly long oval as shown in a phase contrast micrograph (FIG. 4). The biochemical properties of Pseudomonas sp. Of the present invention showed positive reactions in the oxidase test and the catalase test as shown in Table 1 below, esculin, gelatin, and casein. Was hydrolyzed. And the carbon source availability is shown in Table 2 below. Glucose, Arabinose, Mannose, Mannitol, N-Acetyl-Glucosamine, Gluconate except Maltose and Phenyl-Acetate Sugars such as gluconate, caprate, adipate, malate and citrate were used.

Biochemical Properties of Pseudomonas SP C-1 characteristic result Oxidase + Catalase + Reduction of nitrates to nitrites - Indole production - Arginine dihydrolase + β-galactosidase - Lysine decarboxylase - Ornithine decarboxylase - Tryptophane deaminase - Acetoin production - H ₂ S production - Anaerobic growth w Hydrolysis Esulin + Urease - Gelatin + Starch - CM-Cellulose - Casein +

Carbon Source Availability of Pseudomonas SP C-1 Strains characteristic result Glucose + Arabinose + Mannose + Mannitol + N-Acetyl-Glucosamine + Maltose - Gluconate + Caprate + Adipate + Malate + Citrate + Phenyl-acetate -

(2) 16S rRNA sequencing

In order to identify the bacteria, 16S rRNA gene sequencing was used, i.e., the DNA of the isolated strain was extracted with a DNA extraction kit (Toyobo, Japan), followed by the universal primers fD1 (5'-AGAGTTTGATCCTGGCTCAG-3 ') and rP2 (5). 16S rDNA gene was amplified by PCR using '-ACGGCTACCTTGTTACGACTT-3'). The PCR product thus obtained was reacted using a DNA sequencing kit (BigDye terminator Cycle Sequencing Ready Reacions v3.1; Applied Biosystem), and then the base sequence was analyzed by 3100 Genetic Analyser (Applied Biosystems). The sequence was identified to the genus through the BLAST program of NCBI server. To analyze the relationship between strains, the 16S rDNA sequences were aligned with those of the standard strains using the CLUSTAL W program (Thompson et al., 1994). The evolutionary tree of the dataset was created using the MEGA version 3.1 (Kumar et al., 2004) program. The stability of the branch (bootstrap value) was investigated by 1,000 resampling.

As a result of phylogenetic analysis, as shown in FIG. 3, the standard strain having the highest sequence similarity was Pseudomonas. rhodesiae (99.0%), Pseudomonas tolaasii (99.0%), Pseudomonas veronii (99.0%), Pseudomonas marginalis (99.0%), Pseudomonas grimontii (99.2%), Pseudomonas orientali s (99.2%), Pseudomonas meridiana (99.4%), and the relation of genealogy was Pseudomonas was highest with meridiana higher flexibility also related to other Pseudomonas strains sp. The strain according to the present invention was named Pseudomonas SP CL-1.

Example  3: poorly soluble phosphoric acid of the strain Solubilization  And Bacteria

Phosphorus solubilization amount was measured by inoculating Pseudomonas sp. CL-1 strain selected to the culture medium containing calcium phosphate of poorly soluble phosphate. The strains selected were preincubated for 1 day as the inoculum. Already sterilized Pikovskaya's medium: Ca ₃ (PO ₄ ) ₂ 5g, glucose 10g, (NH ₄ ) ₂ SO ₄ 0.5g, KCl 0.2g, MnSO ₄ 0.01g, distilled water 1L, pH7.0 1 ml (1 × 10 ⁷ cfu / ml) of inoculum was added to 100 ml and incubated at 130 rpm at 28 ° C. for 8 days. Sampling of the culture solution was sampled every 24 hours to measure the amount of phosphate solubilization and the number of bacteria. In the analysis of the amount of phosphate solubilization, the cells were separated by centrifugation of the collected culture solution at 10000 × g, and the supernatant was filtered again using a filter of 0.45 μm, and then the amount of phosphate solubilization was measured. The solubilized phosphoric acid was measured by the Banado molybdate method, and the solubilization amount was expressed in mg P / l.

Phosphate Solubilization of Soluble Calcium Triphosphate in the Culture of Pseudomonas SP. Applied Dissolution Rate by Elapsed Days (mg P / ℓ) 0 One 2 3 4 5 6 7 8 CL-1 4.0 ± 0.7 148.0 ± 0.0 154.1 ± 2.9 193.4 ± 1.5 117.5 ± 2.2 14.9 ± 0.0 25.2 ± 0.0 18.5 ± 0.7 22.6 ± 0.7 Control 3.5 ± 0.0 4.5 ± 1.5 3.0 ± 0.7 3.5 ± 0.0 3.5 ± 0.0 3.0 ± 0.7 5.1 ± 0.7 4.0 ± 0.7 3.5 ± 0.0

Increased bacterial counts of Pseudomonas sp. CL-1 in culture Bacterial Growth by Days (cfu / mL) 0 One 3 5 7 CL-1 2.1 × 10 ⁴ 4.7 × 10 ¹⁰ 5.2 × 10 ¹¹ 9.4 × 10 ¹¹ 9.2 × 10 ¹¹ Control 0 0 0 0 0

As shown in Table 3, the strain of the present invention is a strain having an excellent function with a large amount of phosphate solubilizing amount in a short time, and at the same time, the solubilized phosphorus was reused in the microbial composition at a high speed. In other words, the amount of phosphate solubilization increased until 3 days, but on the 5th day, the phosphorus content decreased sharply, while the number of bacteria was the highest, which means that phosphate, an essential component of microbial composition, was reused in microbial composition. Means that. That is, it means that by retaining in the microorganism biologically blocks the fixation to the soil, and also solubilized phosphorus slowly released into the soil in the process of killing the microorganism. Therefore, the phosphoric acid component required by the plant is continuously released during the killing of microorganisms, so the plant growth promoting effect can be expected at the same time.

Example  4.: How to use strain

The method of utilizing the phosphate solubilizing bacterium Pseudomonas sp. CL-1 of the present invention can be used directly in the soil by diluting 20 times or more of the cultured by inoculating TSB (Tryptic Soy Broth) medium.

Increasing Activity in Soil The strain (1 × 10 ⁷ cfu / ml) of the present invention can be inoculated in rice bran and wheat bran, and then grown in 25 ~ 30 ° C. after appropriate amount adjustment to the soil.

In addition, by appropriately adding a nitrogen source, carbon source and inorganic salts can promote the growth of the strain of the present invention, when mixed with the by-product compost mixed with organic material can effectively induce the growth of microorganisms. The amount used may vary depending on the type of crop, the amount of fertilization and the time of use.

The present invention relates to a method for providing Pseudomonas sp. CL-1 strain excellent in solubilizing a poorly soluble phosphate immobilized on soil, and also to a method for solubilizing poorly soluble phosphate. Pseudomonas sp. CL-1 strain solubilizes poorly soluble phosphate and converts it into a form that is readily available to plants. Appropriate use of the strain of the invention reduces phosphate fertilizer and preserves the soil environment. In addition, the strain of the present invention is a strain having a function of excellent phosphate solubilization ability in a short time and reused solubilized phosphorus in the composition of the microorganism at a high speed to block the fixation to the soil, the phosphorus derived from the microorganism in the process of killing the microorganism It is then slowly released into the soil to continuously supply the phosphates required by plants.

<110> Rural Development Administration <120> Pseudomonas sp. CL-1 and Method of solubilizing          insoluble-phosphate by using the same <130> 1 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1410 <212> DNA Pseudomonas sp. CL-1 <400> 1 aacacatgca agtcgagcgg tagagagaag cttgcttctc ttgagagcgg cggacgggtg 60 agtaatgcct aggaatctgc ctggtagtgg gggataacgt tcggaaacga acgctaatac 120 cgcatacgtc ctacgggaga aagcagggga ccttcgggcc ttgcgctatc agatgagcct 180 aggtcggatt agctagttgg tgaggtaatg gctcaccaag gcgacgatcc gtaactggtc 240 tgagaggatg atcagtcaca ctggaactga gacacggtcc agactcctac gggaggcagc 300 agtggggaat attggacaat gggcgaaagc ctgatccagc catgccgcgt gtgtgaagaa 360 ggtcttcgga ttgtaaagca ctttaagttg ggaggaaggg cagttaccta atacgtgatt 420 gttttgacgt taccgacaga ataagcaccg gctaactctg tgccagcagc cgcggtaata 480 cagagggtgc aagcgttaat cggaattact gggcgtaaag cgcgcgtagg tggtttgtta 540 agttggatgt gaaatccccg ggctcaacct gggaactgca ttcaaaactg actgactaga 600 gtatggtaga gggtggtgga atttcctgtg tagcggtgaa atgcgtagat ataggaagga 660 acaccagtgg cgaaggcgac cacctggact aatactgaca ctgaggtgcg aaagcgtggg 720 gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg 780 gaagccttga gcttttagtg gcgcagctaa cgcattaagt tgaccgcctg gggagtacgg 840 ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt 900 ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac atccaatgaa ctttccagag 960 atggattggt gccttcggga acattgagac aggtgctgca tggctgtcgt cagctcgtgt 1020 cgtgagatgt tgggttaagt cccgtaacga gcgcaaccct tgtccttagt taccagcacg 1080 taatggtggg cactctaagg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140 caagtcatca tggcccttac ggcctgggct acacacgtgc tacaatggtc ggtacagagg 1200 gttgccaagc cgcgaggtgg agctaatccc ataaaaccga tcgtagtccg gatcgcagtc 1260 tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc gaatcagaat gtcgcggtga 1320 atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaccagaa 1380 gtagctagtc taaccttcgg gaggacggta 1410  

Claims (3)

Pseudomonas sp. CL-1 (KACC 91282P) strain solubilizing poorly soluble phosphate immobilized in soil. A microbial fertilizer comprising the cells of the Pseudomonas sp. CL-1 strain of claim 1 or a culture medium thereof as an active ingredient. A method of solubilizing poorly soluble phosphate immobilized in soil by using the microbial fertilizer of claim 1 or the microbial fertilizer of claim 2 directly.

Images