KR101377874B1 - Novel Bacillus aryabhattaiLKS28 comprising solubility upon insoluble salts. - Google Patents

Abstract

The present invention relates to a solubilizing insoluble phosphate by treating Bacillus arabhattai LKS28, a novel strain having a solubilizing activity against insoluble salts and biowaste fossils, and this strain on crops and crop growing soils. .
Bacillus arabhattai ( LKS28 ) of the present invention makes soluble phosphate ions, soluble magnesium, calcium ions used by living organisms as wastewater insoluble in phosphate trimium phosphate, tricalcium phosphate as well as biological waste resources. .

Description

Novel Bacillus aryabhattai LKS28 comprising solubility upon insoluble salts.} New strain with solubilizing activity against insoluble salts and fossil fossils.

The present invention relates to a novel strain Bacillus arabhattai LKS28 having solubilizing activity against insoluble salts and pulverized fossils, and solubilizing insoluble salts by treating this strain to crops and crop cultivation soils.

Phosphorus is a very important essential nutrient for plant growth and development. It is a macronutrient that requires a large supply. For maximum crop yield, the availability of phosphorus available to plants is a very important condition. Plant growing soils contain between 400 and 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 is present 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, the supply of inorganic phosphate fertilizers with soluble phosphorus to the soil changes more than 75% of them very quickly to insoluble phosphates, allowing plants to use less than 25% 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 fertilizers 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 by this excessive fertilization is changed from farmland to large insoluble phosphate, which accumulates and eventually becomes salty soils in which plant growth and development are not normally performed.

Moreover, these salt barrier soils occur more frequently in facility cultivation producing high value-added crops, which is a great problem for agricultural productivity and agricultural economy.

Fortunately, it is known that there are microorganisms in the soil that convert these insoluble phosphates into solubilized state. These microorganisms that solubilize these insoluble phosphates (PSBs) are a type of symbiosis that allows the plant to absorb and use soil by converting insoluble phosphates in the soil into solubilized phosphorus by releasing organic acids or secreting specific enzymes in the plant's root zone. It is assumed that the relationship is maintained (Goldstein, Am. J. Altern. Agric. 1986, 1: 51-57).

PSB microorganism Pseudomonas (Pseudomonas), Bacillus (Bacillus), rayijo Away (Rhizobium), Burkholderia (Burkholderia), Agrobacterium (Agrobacterium), micro Cocos (Microccocus), Aero bakteo (Aerobacter), Flavobacterium ( Flavobacterium ) and only a few species belonging to Erwinia .

Examination of these insoluble phosphate solubilization spectra showed overall low activity when only soluble to a specific insoluble phosphate or its broad solubility (lllmer 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 the 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 discover new insoluble phosphate solubilizing microorganisms (PSBs) in addition to known insoluble phosphate solubilizing microorganisms have been reported to allow Rahnella aquatilis to solubilize hydroxyapatite ( Kim et al., FEMS Microbiol. Lett., 1997, 153: 273-277), a 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., Soil Biol. Biochem., 1998, 30 : 995-1003).

Meanwhile, Korean Laid-Open Patent Publication No. 2002-0017516 (a new microorganism that solubilizes phosphate immobilized on volcanic ash soil) discloses about Bacillus sparycus PBS-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 soils.

The recent rapid increase in facility cultivation area has resulted in overaccumulation of salts and nutrients due to the lack of awareness of the farming environment of farmers, resulting in impediment to crop growth, such as imbalance of essential nutrients in soil, water absorption of crops, and salt concentration disturbance. It is emerging as an important problem of facility cultivation. Inorganic salts are an important factor in the growth of crops. The crop absorbs the required amount of salt and does not absorb the rest, so if the microbes do not consume or increase their absorbency, the salt will continue to accumulate and the plant will not survive.
In addition, the amount of shells (shell fossils / shells) and animal bones (bovine bone, pork bone, etc.) belonging to biological waste resources is very high nationwide. Oysters and shells are 255,559 tons (140,750 tons in Gyeongnam, 107,569 tons in Jeonnam, 4,018 tons in Chungnam). Oysters and shells are increasing due to the development of fisheries and aquaculture, but they are mainly landfilled, but the recycling rate is not high. Animal bones are in a similar situation. Therefore, eco-friendly green technology that can increase the value of agricultural food by-products and waste resources (bones, oysters, shells) and recycle at the same time is not only economically important, but also to preserve the global environment and protect human health at home and abroad. very important.

An object of the present invention is a novel Bacillus aryabhattai LKS28 having an insoluble salt solubilizing activity (PS ⁺ ) and a strain that solubilizes insoluble phosphate in crop cultivated soil by treating this strain to crop and crop cultivated soil. To provide.
In addition, it is to provide a strain that makes it possible to recycle biowaste resources by solubilizing the sintered fossil, a biowaste resource, into soluble phosphate ions, soluble magnesium and calcium ions used by living organisms.

According to the present invention, Bacillus arabhattai LKS28 is provided to solubilize insoluble salts in the soil and sintered fossil rocks. This strain is treated on crops and crop cultivated soils to solubilize insoluble salts (3 magnesium phosphate, tricalcium phosphate) and calcite, a bio-waste resource, to make soluble phosphate ions, soluble magnesium, and calcium ions used by living organisms. Enable recycling.

1 is a photograph showing the insoluble phosphate solubilizing ability of Bacillus aryabhattai ( LKS28 ) of the present invention
1: solubilization ability for magnesium triphosphate (Mg ₃ (PO ₄ ) ₂ ), 2: solubility for calcium triphosphate (Ca ₃ (PO ₄ ) ₂ ), 3: solubility for calcite
Figure 2 is a diagram showing the phylogenetic root of Bacillus aryabhattai LKS28 of the present invention
Figure 3 is a graph showing the change in electrical conductivity after inoculating Bacillus aryabhattai ( LKS28 ) salt inoculated in soil
Figure 4 is a graph showing the change in the concentration of phosphate (Pi) ion after inoculating Bacillus aryabhattai ( LKS28 ) in salted soil
5 is a graph showing changes in growth curve (●-●) and phosphate concentration (■-■) of Bacillus aryabhattai LKS28 when insoluble phosphate was supplied to the phosphate source.
Figure 6 Bacillus Ari Abkhatai ( Bacillus aryabhattai ) Effects of Plant Growth on Treatment of LKS28 in Hazardous Soils
A: lettuce, B: cabbage, C: pepper 1: control, 2: LKS2 experimental group
7 is a growth promoting effect when the solubilizing bacteria were mixed treatment (Bisella Kimchi LKS2, Bisella Corriensis LKS42, Lactococcus Lactis LKS49, Bacillus Ariaphatai LKS28)
A: lettuce, B: cabbage, C: pepper, 1: control, 2: mixed treatment (LKS2, LKS42, LKS49, LKS28) experimental group

In order to select strains having high insoluble phosphate solubility, the present inventors have identified microorganisms isolated from kimchi and soil in solid medium containing insoluble phosphates (3 magnesium, tricalcium phosphate, hydroxyapatite, phosphate, phytate), respectively. Strains of high solubility of insoluble phosphate were selected by analyzing the size of the clear ring of solid medium which appeared when inoculated.

The homology of 16S ribosomal DNA obtained from the selected strains was compared and the biological properties were investigated. As a result , a novel strain of Bacillus aryabhattai was identified, which was identified as Bacillus aryabhattai LKS28. The strain was deposited with KCTC 12085BP on November 23, 2011, at the Korea Institute of Bioscience and Biotechnology, an international microbial deposit institution for patent applications.

Bisella Kimchi LKS2, Bisella Corriensis LKS42, and Lactococcus Lactis LKS49, which have been researched and developed by the inventors of the present invention, have similar functions of solubilizing insoluble phosphate in soil, but DNA sequencing and phylogenetic relations A survey of the islands revealed that different genus and other species were.

Bacillus aryabhattai LKS28 of the present invention is a Gram-positive bacterium, and formed colonies of around 4 mm in size in NA medium.

Bacillus aryabhattai ( LKS28 ) of the present invention has a high solubilizing ability of a wide range of insoluble phosphates can be useful for improving the salt impaired soil and plant growth.

In addition, the present invention is the Bacillus Ari Abkhatai ( Bacillus aryabhattai ) Provides a way to improve the impaired soil and improve plant growth using LKS28.

Method for improving the soil disturbance by using the strain of the present invention and promote the plant growth, the tryptophan-Ari Bacillus reduction in the yeast broth or MRS broth tie (Bacillus aryabhattai ) and the first step of culturing the LKS28, and Bacillus aryabhattai ( Bacillus aryabhattai ) LKS28 is inoculated into salty soil soil and the second step to enhance the growth of the plant.

Incubate for 1 to 2 days at 30 ~ 36 ℃ during the culture of the first step.

Cultured Bacillus Ariaphathai aryabhattai ) KLS28 is washed with distilled water or isotonic solution of 0.8% NaCl.

Stored Bacillus Ariaphathai aryabhattai ) Inoculate the LKS28 at 1X10 ⁷ CFU / ml twice a week in saline soils, and then grow the germinated crops from the soil.

Bacillus Ariaphatai of the present invention for four more weeks at intervals of one week after crop transplantation (Bacillus aryabhattaiInoculate LKS28.

Bacillus Ariabhatai of the present invention by the above method ( Bacillus aryabhattai ) The inoculation of LKS28 results in the normal growth and development of crops even in saltwater soils.

Hereinafter, the present invention will be described in detail by way of examples, but the contents of the present invention are not limited thereto.

< Example  1> Selection of Strains

In order to select strains from the soil, the soil collected in Korea was used as a microbial extraction source by filtering 2mm 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 stirred 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.

For the selection of strains from Kimchi, the cells were cultured in MRS medium and tryptone yeast (TYE) medium, and then separated and selected.

  Microorganisms isolated from soil and kimchi were inoculated and incubated in solid medium containing insoluble phosphate (3 magnesium phosphate, tricalcium phosphate, calcite) in MOPS medium, which is the minimum medium.

After incubation, the insoluble phosphate solubilizing strain showing a clear ring in a solid medium was pure culture.

Pure cultured strains were inoculated in the above insoluble phosphate solid medium, respectively, to investigate the insoluble phosphate solubilization range of each strain (FIG. 1).

The size of the transparent ring was analyzed to isolate strains with high solubility of insoluble phosphate.

< Example 2 > Investigation of genetic similarity between nucleotide sequence of isolated strain and existing strain

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

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

또한 이 염기서열을 NCBI data bank의 유전정보를 다음과 같이 비교분석한 결과 Bacillus aryabhattai 16S ribosomal RNA gene과 100％의 유사도를 보이고 있음을 확인하였다.

The determined 16S rDNA nucleotide sequence was compared with the strains registered in the NCBI gene bank to investigate the phylogeny and genetic similarity, and the results are shown in FIG.
The base sequence of 16S rDNA of Bacillus aryabhattai LKS28 , which is selected strain, is as follows.

In addition, this sequence was compared with the genetic information of the NCBI data bank as shown below, and showed 100% similarity with Bacillus aryabhattai 16S ribosomal RNA gene.

As shown in Figure 3, the selected strain is Bacillus Ariaphatai ( Bacillus aryabhattai ).

The present inventors Ari reduction Bacillus Thai (Bacillus a starter strain aryabhattai ) It was named LKS28 and it was registered and deposited with Korea Institute of Biotechnology and Biotechnology Center on November 23, 2011. (Accession No.: KCTC 12085BP)

< Example  3> Bacillus Ariaphathai  ( Bacillus aryabhattai ) LKS28 Utilization of strain

Prepare tryptone-yeast (TYE) liquid medium.

Bacillus Ariaphatai obtained in Example 1 ( Bacillus aryabhattai ) LKS28 was incubated at 36 ° C for 2 days.

Cultured Bacillus Ariaphathai aryabhattai ) LKS28 microorganisms were stored by washing with isotonic solution of NaCl 0.8%.

Prepared Bacillus Ariaphathai aryabhattai ) LKS28 was salted and diluted in distilled water at a concentration of 1 × 10 ⁷ CFU / ml in soil and inoculated twice a week.

After transplanting the germinated crops from the topsoil, the inoculation was further inoculated once a week for 4 weeks, and the soil was improved by utilizing the microorganism of the present invention in the crops in the salt impaired soil.

< Example  4> Improved salt disorder soil

Bacillus Ari Abkhatai of the present invention ( Bacillus aryabhattai ) LKS28 was incubated at 30 ° C. for 2 days, and then Bacillus aryabhattai LKS28 was diluted in 50 ml of distilled water at a concentration of 1 × 10 ⁷ CFU / ml, and then inoculated into 150 g of saline-injured soil. The change was investigated.

The control was treated only with distilled water.

The soil solution for measuring the electrical conductivity was used after distilled water was added for 2 to 3 hours so that the ratio of soil and water 1: 5, and then filtered and leached.

As a result, the electrical conductivity of the salt disturbed soil before inoculation was 10.2 ds / m gradually lowered after inoculation, and after 4 days, it was 6.0 ds / m or less (see FIG. 3).

From the above results, Bacillus aridhathai of the present invention ( Bacillus aryabhattai ) LKS28 strain was found to have an effect of reducing the electrical conductivity of salt soil soil.

< Example  5> Solubilization  Investigation of phosphorus content change

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 molybdenum blue method with ascorbic acid.

In the experimental group, Bacillus aryabhattai ) Diluted LKS28 in 50ml of distilled water at the concentration of 1X10 ⁷ CFU / ml, inoculated into 150g saline soil, take 10g of treated soil every 24 hours, and add 50ml of distilled water as in the control group for 3 hours. After shaking, the mixture was filtered and used as a soil solution.

The measurement of phosphorus was also measured using molybdenum blue method with ascorbic acid.

As a result, the content of phosphorus solubilized in salt-impacted soil was 25 ㎍ / g, compared to Bacillus Inoculated with LKS28 strain, Bacillus arihathatai of the present invention ( Bacillus) of the present invention from the above results formed three times more water-soluble solubilized phosphorus than the control group for 4 days after treatment aryabhattai ) LKS28 strain was found to be a biofertilizer bacteria (biofertilizer) to produce a large amount of water-soluble solubilized phosphorus from salt sea soil (Fig. 4).

< Example  6> various insoluble phosphate Availability and  Growth rate and Salt removal ability

On the other hand, after depleting phosphoric acid in the MOPS medium, which is the smallest medium, insoluble phosphate tricalciumphosphate and trimagnesiumphosphate were added to the phosphate solubilizing medium at 0.5%, respectively, to prepare the medium, and then inoculated with the strains. 10 ml of each was measured, and the growth rate was measured. After centrifugation of the culture solution, the solubilized Pi in the medium was measured using the supernatant, and the solubilization and degradation of insoluble phosphate were examined.

As a result, the growth rate was slightly different between insoluble phosphate medium, but most of the strains showed maximum growth at 3 days after incubation. In the case of the free Pi content change, the amount of Pi was high in both the three calcium phosphate medium and the three magnesium phosphate medium supplied as the only personnel, indicating that the solubilization of tricalcium phosphate and magnesium phosphate was very vigorous (FIG. 5).

< Example  7> Changes in germination rate of crops

In order to determine the change in germination rate of crops for each strain, incubate in TYE medium for 1 day, wash three times with 0.85% saline solution, suspend in sterile distilled water, and apply two layers of Whatman No 2 filter paper to four petri dishes. After spreading on the bottom, each strain was treated to 1 × 10 ⁷ CFU / ml. After inoculating 5 ml of suspension, 25 seeds of Chinese cabbage were placed on a plate, and the germination rate was examined after 2 and 3 days. Sterile distilled water was used as a control. As a result, there was no significant difference compared to the control group. Rather, when the phosphate solubilized strains detected by the researchers were treated, the germination rate was the same on the 3rd day compared to the control, or the germination rate increased by 2% for LKS42 and LKS49. Could be (see Table 1.). As a result, the insoluble phosphate solubilized strains searched by the researchers did not cause any damage to the seeds.

Table 1. Changes in germination rate of Chinese cabbage DW LKS2 Suspension LKS42 Suspension LKS49 Suspension LKS28 Suspension 96 96 98 98 96

< Example  8> Pott Experiment

Based on the results of the germination rate in Table 1, each seed (lettuce, cabbage, red pepper) is germinated in the top soil, and when two leaves are emerged, it is formulated in a pot filled with obstacle soil, and Bacillus ariphatai ( Bacillus) aryabhattai ) LTS28 was treated to 10 ⁷ CFU / ml before pot experiments were performed. After inoculation, the insoluble phosphate solubilizing strain was inoculated to examine the growth rate at 7 days. As a result, the growth rate was relatively excellent when each strain was inoculated into the obstacle soil as compared to the obstacle soil (control) (Fig. 6).

On the other hand, when comparing the growth rate when the solubilized strains were applied to the mixed soil mixed with the simultaneous patent application, it was excellent in each crop compared to the control (Fig. 7).

Claims (6)

Bacillus aryabhattai LBS28 (Accession No .: KCTC 12085BP) Method of inoculating the strain of claim 1 into a crop or crop cultivating soil to solubilize insoluble salts in the soil to promote soil improvement and crop growth. Method for solubilizing crushed fossil stone as a biological waste resource using the strain of claim 1 delete delete delete

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