KR100460634B1 - The novel enterobacter intermedius ksj16 which solves insouble phosphate in soil - Google Patents

Abstract

The present invention relates to a novel strain Enterobacter intermedius KSJ16 that solubilizes insoluble phosphate and to solubilizing insoluble phosphate by treating this strain with crop cultivated soil.

Enterobacter intermedius KSJ16 of the present invention makes insoluble phosphate triphosphate, hydroxyapatite, phosphate, phosphate, and the like in the soil into soluble phosphate ions used by living organisms.

Description

New strain Enterobacter intermedius KJJ that solubilizes insoluble phosphate in soil {THE NOVEL ENTEROBACTER INTERMEDIUS KSJ16 WHICH SOLVES INSOUBLE PHOSPHATE IN SOIL}

The present invention relates to a novel strain Enterobacter intermedius KSJ16 that solubilizes insoluble phosphate, and to a method of solubilizing insoluble phosphate by treating this strain to crop cultivated soil.

Phosphorus is a very important essential nutrient for plant growth and development. It is a macronutrient that requires a large supply. Therefore, for the maximum harvest of major crops in agriculture, the availability of phosphorus available to plants is a very important condition. Plant growing and developing soils contain 400 to 1200 mg / kg of phosphorus (Rodriguez and Fraga, Biotech. Adv . 1999, 17: 319-339).

However, most of the phosphorus in the soil is insoluble, so plants cannot use it. In other words, phosphorus in the soil exists in the form of chemically tightly bound insoluble complexes or organically bound phytates and insoluble phosphates that cannot be used by plants (Paul and Clark, Soil Microbiology and Biochemistry). , Academic Press, San Diego, CA, 1996).

Thus, available phosphorus in natural soils is present in less than the concentration of micronurtients. After all, the concentration of soluble phosphorus is a limiting factor in the promotion of plant growth and development in the soil. Moreover, even when the inorganic phosphate fertilizer containing soluble phosphorus is supplied to the soil, more than 75% of them change very quickly to insoluble phosphate state, so that plants can use only 25% or less of the soluble phosphorus supplied (Goldstein, Am. J. Altern. Agric. 1986, 1: 51-57).

Therefore, farmers continue to repeat the excessive fertilization of excessive supply of inorganic phosphate fertilizer to the farmland in order to prevent the deterioration of plant growth and development due to the lack of soluble phosphorus. However, most of the soluble phosphorus supplied at such excessive fertilization changes from farmland to large amounts of insoluble phosphate, which accumulates, eventually resulting in salt-damaged soils in which plant growth and development are not normally performed.

In addition, these salt-impacted soils occur more frequently in facility cultivation as a system for producing high value-added crops, which is a great problem for agricultural productivity and agricultural economy.

Fortunately, it is known that there are some microorganisms in the soil that can convert these insoluble phosphates into solubilized state. These insoluble phosphate solubilizing bacteria (PSBs) maintain a kind of symbiotic relationship by releasing organic acids or secreting specific enzymes in the root zone of plants to convert insoluble phosphates in the soil into solubilized phosphorus, allowing the plant to absorb and use them. (Goldstein, Am. J. Altern. Agric. 1986, 1: 51-57).

PSB microorganisms include Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Agrobacterium, Microccocus and Aeoleobacter. (Aereobacter), Flavoacterium and Erwinia.

Examination of their insoluble phosphate soluble spectra showed overall low activity when only soluble to a specific insoluble phosphate or a wide range of solubility (Illmer and Schinner, Soil Biol. Biochem., 1992, 24: 389-395; Rodriguez et al., Rev. ICIDCA,1996, 30: 47-54; Arora and Gaur, Indian J. Exp. Biol.,1979, 17: 1258-1261; Halder and Chakrabartty, Folia Microbiol.,1993, 38: 325-330).

PSB microorganisms are present in the soil but are not always high enough to compete with other microorganisms that form the plant root zone. Thus, the amount of soluble phosphorus produced by PSB microorganisms in the soil by breaking down insoluble phosphates is not high enough to increase plant growth and development. Therefore, artificially inoculating large amounts of PSB microorganisms into the soil has the potential to increase plant growth and development.

This research has been reported to improve plant growth using insoluble phosphate solubilizing microorganisms (Kloepper et al., ISI Atlas Sci. Anim. Plant Sci.,1988, pp. 60-64; Chabot et al., Appl. Environ. Microbiol., 1996, 62: 2767-2772).

Efforts to find new PSB microbes in addition to the known PSB microorganisms have been raced to report that Rahnella aquatilis can solubilize hydroxyapatite (Kim et al., FEMS Microbiol). Lett., 1997, 153: 273-277), the related gene pqq has also been identified (Kim et al., FEMS Microbiol. Lett., 1998, 159: 121-127).

Kim et al. Also confirmed that Enterobacter agglomerans can also produce soluble phosphorus by decomposing hydroxyapatite (Kim et al., Soil Biol. Biochem., 1998, 30). : 995-1003).

On the other hand, Korean Laid-Open Patent Publication No. 2002-0017516 (a new microorganism that solubilizes phosphate immobilized on volcanic ash soil) discloses about Bacillus sparycus PSB-13 solubilizing phosphate immobilized on volcanic ash soil.

However, there is still a need to develop new strains that solubilize insoluble phosphate in crop cultivated soil.

It is an object of the present invention to provide a novel microbial strain that solubilizes insoluble phosphate in crop cultivation soil with higher efficiency.

It is also an object of the present invention to provide a method of solubilizing insoluble phosphate in crop cultivation soil using a novel strain.

1 is a photograph showing the insoluble phosphate solubilizing ability of Enterobacter intermedius KSJ16 of the present invention.

Above: Solubilization capacity for hydroxyapatite.

Bottom: Solubility on calcium triphosphate.

Figure 2 is a diagram showing the phylogenetic root of the Enterobacter intermedius KSJ16 of the present invention.

Figure 3 is a graph showing the change in electrical conductivity after inoculation of Enterobacter intermedius KSJ16 in saltwater soil.

Figure 4 is a graph showing the change in the concentration of phosphorus (Pi) after inoculating Enterobacter intermedius KSJ16 to salt sea soil.

Figure 5 is a photograph showing the growth of the plant as a result of soil treatment with Enterobacter intermedius KSJ16.

A: Picture of cultured lettuce B: Picture of cultured cabbage

Control: Untreated with Enterobacter intermedius KSJ16.

KSJ8: Inoculated with Enterobacter intermedius KSJ16.

The present invention relates to a novel strain Enterobacter intermedius KSJ16 that solubilizes insoluble phosphate, and to a method of solubilizing insoluble phosphate by treating this strain to crop cultivated soil.

Enterobacter intermedius KSJ16 of the present invention makes insoluble phosphate tricalcium hydroxyapatite, hydroxyapatite, phosphate, phytate, and the like in the soil into soluble phosphate ions used by living organisms to enhance plant growth.

Hereinafter, the present invention will be described in detail.

In the present invention, in order to select a strain having high insoluble phosphate solubility, the transparent ring of the solid medium that appears when the microorganism isolated from the soil is inoculated into a solid medium containing insoluble phosphates (calcium triphosphate and hydroxyapatite), respectively, and cultured. The size was analyzed to select strains with high solubility of insoluble phosphate.

The homology of 16S ribosomal DNA obtained from the selected strains was compared and the biological properties were investigated. As a result, it was found to be a novel strain of Enterobacter intermedius , which is known as Enterobacter intermedius . The strain was named KSJ16, and the strain was deposited with the accession number KCTC 10387BP on November 28, 2002 to the Korea Biotechnology Research Institute Gene Bank, an international microbial deposit institution for patent applications.

The Leclercia adecarboxylata KSJ8, which has been studied and developed by the inventors of the present invention, has a similar function to solubilize the insoluble phosphate in soil and its physiological and biochemical properties, but is similar to the DNA sequence and phylogenetics. Investigating the recent years, it was found that different genus and other species.

Enterobacter intermedius KSJ16 of the present invention is Gram-negative bacteria, rod-shaped, and motility.

Enterobacter intermedius KSJ16 of the present invention hydrolyzes esculin and gelatin, and hydrolyzes tryptophan to produce indole, catalase and oxidase ) Is also active. In addition, citrate, phenylalanine and lactose can be used, and organic acids are produced by glucose fermentation.

From the above results, Enterobacter intermedius KSJ16 of the present invention has high solubility of a wide range of insoluble phosphates, and thus may be useful for improving salt disorder soil and enhancing plant growth.

In addition, the present invention provides a method for improving impaired soil improvement and plant growth using the Enterobacter intermedius KSJ16.

Method for improving the impaired soil and improve plant growth using the strain of the present invention, the first step of culturing Enterobacter intermedius KSJ16 in tryptophan-east (TYE) liquid medium, and cultured Enterobacter intermedius It consists of the second step of inoculating KSJ16 into salty soils to promote plant growth.

Incubate for 2 days at 25 ~ 30 ℃ in the first step of culture.

The cultured Enterobacter intermedius KSJ16 is stored by washing with distilled water or isotonic solution of 0.8% NaCl.

After inoculation of the stored Enterobacter intermedius KSJ16 at 1x10 ⁷ cfu / ml, twice a week, in salt sea soil, the crops germinated in the soil are transplanted to grow the crops.

After the plant transplantation, the bacterium Enterobacter intermedius KSJ16 of the present invention is further inoculated for 4 weeks at weekly intervals.

When inoculated Enterobacter intermedius KSJ16 of the present invention by the above method, the growth and development of normal crops occurs even in saltwater soil (see FIG. 5).

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the content of the present invention is not limited thereto.

Example 1 Selection of Strains

Soil collected in Korea was used as a microbial extraction source by filtering 2 mm sieve.

50 ml of 25% Ringer's solution was added to 5 g of wet soil, shaken for 2 minutes, and stirred for 1 hour.

The agitated soil sample was repeated three times for 5 minutes sonication was removed from the soil particles.

After centrifugation at 1,500 rpm for 10 minutes, the supernatant was used as a screening sample for insoluble phosphate solubilizing microorganisms (PSB).

The isolated microorganisms were inoculated and incubated in a solid medium containing insoluble phosphate (calcium phosphate, hydroxyapatite) as a material of phosphorus in MOPS medium, which is a minimal medium.

After incubation, insoluble phosphate solubilizing microorganism strains showing transparent rings in solid medium were purely cultured.

Pure cultured strains were inoculated in the above insoluble phosphate solid medium, and the insoluble phosphate solubilization range of each strain was examined.

The size of the clear ring was analyzed to select strains having high solubility of insoluble phosphate (see FIG. 1).

<Experiment 1> Investigation of genetic similarity between the nucleotide sequence of the isolated strain and the existing strain

Genomic DNA of the strain selected in Example 1 was isolated using CTAB solution.

16S ribosomal DNA (rDNA) was amplified and cloned using PCR to determine its nucleotide sequence.

As a result, the nucleotide sequence of this strain appeared as SEQ ID NO: 1 in the attached sequence list.

The nucleotide sequence of the determined 16S rDNA was compared with the strains registered in the NCBI gene bank to investigate the phylogenetic tree and genetic similarity and the results are shown in FIG. 2.

We named the selected strain Enterobacter intermedius KSJ16, and deposited it on November 28, 2002 with the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10387BP).

Experimental Example 2 Efficacy Test of Enterobacter Intermedius KSJ16 Strain

For the Enterobacter intermedius KSJ16 of the present invention, Benson (1990, Microbiological Applications: A Laboratory Manual in General Microbiology, WCB Publishers, Dubuque) and the Korean Society for Microbiology (1998, Microbiology Laboratory , Korean Society for Microbiology, Eulyu Cultural History) Physiological and biochemical properties were investigated according to the method.

As a result, Enterobacter intermedius KSJ16 of the present invention was a motility bacterium, had an esculinase activity that degrades esculin, and had a catalase activity that converts harmful hydrogen peroxide. .

In addition, citrate (citrate) can be used as a carbon source, gelatin (gelatin) has a protein resolution, such as hydrolysis, tryptophan (tryptophan) can be used as an amino acid material to produce indole (indole).

There was also an activity of cytochrome oxidase, which proved to be an aerobic bacterium, and the activity of phenylalanase, which degrades phenylalanine to produce phenylpyruvic acid, There was also activity of beta-galactosidase, which degrades lactose.

In addition, it was confirmed through the methyl red test that the strain fermenting glucose to produce an organic acid.

However, the Bogus-Proskauer test did not produce acetyl-methyl-carbinol (acetoin) or 2,3-butandiole by fermenting glucose, and amylase activity, which degrades starch, It was confirmed that none.

In addition, it was confirmed that the Enterobacter intermedius KSJ16 of the present invention is a rod-shaped Gram-negative bacterium.

Example 2 Application of Enterobacter Intermedius KSJ16 Strain

Tryptophan-yeast (TYE) liquid media was purchased commercially and prepared.

Enterobacter intermedius KSJ16 obtained in Example 1 was incubated at 25-30 ° C for 2 days.

The cultured Enterobacter intermedius KSJ16 strain was stored by washing with isotonic solution of 0.8% NaCl.

Prepared Enterobacter intermedius KSJ16 was inoculated twice a week at a concentration of 1 × 10 ⁷ cfu / ml in saline soil.

After transplanting the germinated crops from the topsoil, once a week for 4 weeks,

The soil was improved by utilizing the microorganisms of the present invention in crops in saline soils.

Experimental Example 3 Salt Impaired Soil Improvement and Plant Growth Enhancement

Enterobacter intermedius KSJ16 of the present invention was incubated at 30 ° C. for 2 days, and then diluted in 50 ml of distilled water at a concentration of 1 × 10 ⁷ cfu / ml in saline soils, and then inoculated in 150 g saline soils. Was inoculated to investigate the change in conductivity.

The control was treated only with distilled water.

The soil solution for measuring the electrical conductivity was used by distilled water and left for 2-3 hours after the ratio of soil and water to 1: 5, filtered and leached.

As a result, the salt conductivity of the soil was 11 ds / m, whereas after inoculation of Enterobacter intermedius KSJ16 was gradually lowered to less than 7 ds / m after 5 days (see Figure 3).

From the above results, it can be seen that the Enterobacter intermedius KSJ16 strain of the present invention has an improvement effect of lowering the electrical conductivity of the salt disturbed soil.

Experimental Example 4 Investigation of Changes in Soluble Phosphorus

In order to investigate the content of water-soluble solubilized phosphorus in salt barrier soil, 50 ml of distilled water was added to 10 g salt barrier soil, shaken for 3 hours, and then used as a soil solution.

The amount of phosphoric acid was measured using molybten blue method with ascorbic acid.

In the experimental group, enterobacter intermedius KSJ16 was diluted in 50 ml of distilled water at a concentration of 1x10 ⁷ cfu / ml, inoculated into 150 g of saline soil, and 10 g of treated soil was taken every 24 hours. After distilled water was shaken for 3 hours, the mixture was filtered and used as a soil solution. The measurement of phosphorus was also measured using molybdenum clarification method with ascorbic acid.

As a result, the content of solubilized phosphorus in the salt-impacted soil was 25 ㎍ / g, whereas when inoculated with Enterobacter intermedius KSJ16 strain, water-soluble solubilized phosphorus was formed more than twice as much as in the control group for 4 days and 28 ㎍ on the 5th day. lowered to / g. However, it increased rapidly from day 6 and confirmed that it contains 239 μg / g of water-soluble solubilized phosphorus on day 8 (see Fig. 4).

From the above results, the Enterobacter intermedius KSJ16 strain of the present invention was found to be a biofertilizer bacteria (biofertilizer) to produce a large amount of solubilized phosphorus from salt sea soil.

Experimental Example 5 Crop Growth Enhancement Effect of Enterobacter Intermedius KSJ16 Strain

Crop growth promoting effect of Enterobacter intermedius KSJ16 strain of the present invention was investigated through pot experiments on lettuce, cabbage and pepper.

Seeds of lettuce, cabbage and red pepper were germinated in the soil, respectively.

Then, when two main leaves came out, they were transplanted into saline soils contained in a Schiropol container 55 cm long, 45 cm wide, and 15 cm high.

Herein, the Enterobacter intermedius KSJ16 strain of the present invention was treated once a week.

The used Enterobacter intermedius KSJ16 strain was cultured in 100 ml of tryptophan-yeast (TYE) for 18 hours, collected by centrifugation, washed twice with distilled water, and suspended in 200 ml of distilled water for one treatment. .

Lettuce was harvested after 3 treatments and cabbage was harvested after 4 treatments.

One week after the last treatment, the growth and development of the crops were examined.

Sufficient water was supplied once a day for the cultivation of crops and no treatment with fertilizers or pesticides. The control group treated only distilled water at the same time.

As a result, when inoculated into the Enterobacter intermedius KSJ16 strain of the present invention compared to the control transplanted into the salt soil soil, it was confirmed that the growth and development of crops clearly distinguished by the naked eye. (See Figure 5).

Total weight, root weight, number of leaves, height, leaf area, and chlorophyll of Chinese cabbage and lettuce were compared as shown in Table 1.

<Table 1> Growth effect of crops

crops division Live weight (g) Root weight (g) The side tree (the head) Extra long (cm) Side area (cm ² ) chlorophyll cabbage Control 189.35 2.20 17 32.5 2512.70 34.12 KSJ16 515.59 4.29 27 40.0 6144.64 37.61 Lettuce Control 17.38 2.47 13 13.7 428.70 31.44 KSJ16 38.07 5.04 16 20.1 880.72 31.17

As shown in Table 1, the crops treated with Enterobacter intermedius KSJ16 strains had more than double the weight of the roots compared to the control cultured in saltwater soils. gave.

In particular, the cabbage was more effective in overcoming the salt damage by Enterobacter intermedius KSJ16 strain.

From the above results, it can be seen that the Enterobacter intermedius KSJ16 strain of the present invention is a plant growth promoting myobacterial bacteria (PGPR) that promotes the growth and growth of crops in saltwater soils.

The present invention provides an Enterobacter intermedius KSJ16 strain that solubilizes insoluble phosphate in soil.

Also provided is a method of promoting plant growth by treating the soil of the present invention with soil.

<110> LEE, Ki-Sung <120> THE NOVEL ENTEROBACTER INTERMEDIUS KSJ16 WHICH SOLVES INSOUBLE PHOSPHATE IN SOIL <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1349 <212> DNA <213> Enterobacter intermedius KSJ16 < 400> 1 caggcctaac acatgcaagt cgaacggtag cacagagagc ttgctctcgg gtgacgagtg 60 gcggacgggt gagtaatgtc tgggaaactg cccgatggag ggggataact actggaaacg 120 gtagctaata ccgcataacg tcgcaagacc aaagtggggg accttcgggc ctcacaccat 180 cggatgtgcc cagatgggat tagctagtag gtggggtaat ggctcaccta ggcgacgatc 240 cctagctggt ctgagaggat gaccagccac actggaactg agacacggtc cagactccta 300 cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag ccatgccgcg 360 tgtatgaaga aggccttcgg gttgtaaagt actttcagcg gggaggaagg cggtggagtt 420 aatagcttca ccgattgacg ttacccgcag aagaagcacc ggctaactcc gtgccagcag 480 ccgcggtaat acggagggtg cgagcgttaa tcggaattac tgggcgtaaa gcgcacgcag 540 gcggtctgtc aagtcccgggga aact 600 ggcaggctgg agtcttgtag aggggggtag aattccaggt gtagcggtga aatgcgtaga 660 gatctggagg aataccggtg gcgaaggcgg ccccctggac aaagactgac gctcaggtgc 720 gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgtcg 780 acttggaggt tgttcccttg aggagtggct tccggagcta acgcgttaag tcgaccgcct 840 ggggagtacg gccgcaaggt taaaactcaa atgaattgac gggggcccgc acaagcggtg 900 gagcatgtgg tttaattcga tgcaacgcga agaaccttac ctactcttga catccagaga 960 acttagcaga gatgctttgg tgccttcggg aactctgaga caggtgctgc atggctgtcg 1020 tcagctcgtg ttgtgaaatg ttgggttaag tcccgcaacg agcgcaaccc ttatcctttg 1080 ttgccagcgg ttcggccggg aactcaaagg agactgccag tgataaactg gaggaaggtg 1140 gggatgacgt caagtcatca tggcccttac gagtagggct acacacgtgc tacaatggcg 1200 catacaaaga gaagcgacct cgcgagagca agcggacctc ataaagtgcg tcgtagtccg 1260 gatcggagtc tgcaactcga ctccgtgaag tcggaatcgc tagtaatcgt agatcagaat 1320 gctacggtga atacgttccc gggccttgt 1349

Claims (2)

Strain Enterobacter intermedius KSJ16 (KCTC 10387BP) solubilizing insoluble phosphate. A method of promoting the growth of crops by inoculating the strain of claim 1 into crop growing soil solubilizing insoluble phosphate in the soil.