KR102095659B1 - AST-4 promoting plant growth under salt condition and uses thereof - Google Patents

Abstract

The present invention relates to a Bacillus cereus AST-4 strain promoting plant growth in a salt stress environment, and to a use thereof.

Description

AST-4 strain and its use in Bacillus cereus, which promotes plant growth in a salt stress environment {AST-4 promoting plant growth under salt condition and uses thereof}

The present invention relates to an AST-4 strain of the genus Bacillus cereus and its use to promote plant growth in a salt stress environment.

Although Korea has a large population, more than 60% of the country's land is mountainous, so it is difficult to supply the necessary land for new industrial complexes, housing complexes, and ports required for economic development. Accordingly, a project for supplying these papers by reclaiming a reclamation land or the sea for supplying necessary papers has been promoted. The land use plan of domestic landfills was primarily aimed at creating industrial facilities in the early days of landfill, but recently, many landscaping trees have been planted for new awareness of the environment and a comfortable living environment.

Landfills or landfills usually use heavy equipment to cut the mountain and collect the soil to reclaim the sea, or to pump dredged materials such as sand, dredged soil, construction residue, garbage or waste, and mountain soil from the sea outside the planned landfill site. do. However, the soil buried with the above materials lacks effective subsoil, excessive moisture due to poor drainage, subsidence of soil, poor structure of soil, lack of soil moisture, irregularity of particle size, lack of plant nutrients, and presence of salt (drinking soil 0.73 ds / m soil 0.36 ds / m range), heterogeneity of pH (pH 7.5 for dredged soil, pH 6.6 for soil), low organic matter (OM 0,5 ~ 1.0), etc., which are not suitable for normal tree growth. Have In addition, the soil has a biological property that the microorganisms are insufficient for the growth of plants such as trees because the salt is excessive and the microorganisms are not suitable for survival.

Accordingly, in order to use the reclaimed land as an agricultural land, the development of a salt-resistant cultivation method of crops has been developed. Rather than develop flame-retardant crops by genetic recombination technology (GMO), many researchers are developing techniques to improve the flame-resistance of crops by applying microorganisms that help plants grow in a salt-stressed environment. For example, Korean Patent Registration No. 10-1563349 discloses a mixture of dendritic mycorrhizal fungi and a genus of genus RK4 that promote plant growth under salt stress conditions and uses thereof, and Korean Patent No. 10-0968382 A strain of the genus Osobacillus having excellent salt resistance and its use are disclosed.

Accordingly, the present inventors studied a strain that promotes the growth of plants in a salt stress environment, and isolates the new strain AST-4 from soil near the vinyl house, and as a result of identification, the strain belongs to Bacillus cereus (Bacillus cereus). Was confirmed. Accordingly, as a result of the AST-4 strain growing on barley, it was confirmed that the content of chlorophyll increased and the present invention was completed.

The present invention was devised to solve the above problems, and thus the present inventor separated a new strain AST-4 (KCTC18752P) from soil near the greenhouse while studying a strain that promotes the growth of plants in a salt stress environment. , As a result of identification, it was confirmed that the strain belongs to Bacillus cereus (Bacillus cereus). Accordingly, as a result of the AST-4 strain growing on barley, it was confirmed that the content of chlorophyll increased and the present invention was completed.

An object of the present invention is to provide a Bacillus cereus (Bacillus cereus) AST-4 (KCTC18752P) having flame resistance.

Another object of the present invention is to provide a microorganism preparation for promoting plant growth in a salt stress environment including the Bacillus cereus AST-4 (KCTC18752P), a culture thereof, or a mixture thereof.

Another object of the present invention is to provide a microbial fertilizer for promoting plant growth in a salt stress environment comprising the Bacillus cereus AST-4, a culture thereof, or a mixture thereof.

Another object of the present invention is to provide a method for promoting plant growth comprising the step of treating the microbial fertilizer to a plant or a plant seed.

Another object of the present invention is to provide a soil conditioner comprising Bacillus cereus AST-4, a culture thereof, or a mixture thereof.

Another object of the present invention is to provide a method for improving soil in a reclamation site, a sea landfill, a vinyl house, or a salt collection site by using the soil improving agent.

The present invention can provide Bacillus cereus AST-4 (KCTC18752P) having flame resistance.

The present invention can provide a microbial agent for promoting plant growth in a salt stress environment, including the Bacillus cereus AST-4, a culture thereof, or a mixture thereof.

The present invention can provide a microorganism fertilizer for promoting plant growth in a salt stress environment, including the Bacillus cereus AST-4, a culture thereof, or a mixture thereof.

The present invention provides a method for promoting plant growth comprising the step of treating the microbial fertilizer to a plant or a plant seed.

The present invention can provide a soil conditioner comprising Bacillus cereus AST-4.

The present invention can provide a method of improving soil in a reclamation area, a sea landfill, a vinyl house, or a salt collection site by using the soil improving agent.

Bacillus cereus genus AST-4 of the present invention has an effect of increasing the content of chlorophyll when treated on nutrient barley in a salt stress environment. In the control group treated with 5% final NaCl concentration in the soil, the total content of chlorophyll in the control barley was 1.51 mg / g, whereas in the experimental group inoculated with the strain Bacillus cereus AST-4, the final NaCl concentration in the soil was treated with 5%. The total content of was 8.18 mg / g, which was 5.42 times higher than that of the control group.

1 is a schematic diagram of the salt-resistant strain AST-4 of the present invention.
Figure 2 is a result showing the change in the chlorophyll content of the leaves and untreated leaves of the barley treated with the strain AST-4 of the present invention in a salt stress environment.

The present invention will be described in detail below.

Although Korea has a large population, more than 60% of the country's land is mountainous, so it is difficult to supply the necessary land for new industrial complexes, housing complexes, and ports required for economic development. Accordingly, a project for supplying these papers by reclaiming a reclamation land or the sea for supplying necessary papers has been promoted. However, land reclamation and land reclamation have excessive salt and are not suitable for microorganisms to survive, and thus have biological properties such as lack of effective microorganisms for plants such as trees to grow. Accordingly, the present inventors studied a strain that promotes the growth of plants in a salt stress environment, and isolates the new strain AST-4 from soil near the vinyl house, and as a result of identification, the strain belongs to Bacillus cereus (Bacillus cereus). Was confirmed. Accordingly, when the AST-4 strain was used to test the growth of barley, the chlorophyll content was increased when the strain was treated on barley, and the present invention was completed.

The present invention can provide Bacillus cereus AST-4 (KCTC18752P) having flame resistance.

The present inventors isolated the new strain AST-4 from the soil near the vinyl house, and as a result of identifying the new strain, it was confirmed that the new strain AST-4 belongs to Bacillus cereus (FIG. 1). The total chlorophyll content of nutrient barley without NaCl treatment in the soil was 17.18 mg / g, which was 11.38 times higher than the total content of chlorophyll nutrient in the control group treated with 5% final NaCl concentration in the soil. The total content of chlorophyll in nutrients of the experimental group inoculated with the strain Bacillus cereus AST-4 after treating the final NaCl concentration of 5% was 8.18 mg / g, which was 5.42 times higher than the control group without the strain. Therefore, the increase of salt in the soil reduces the chlorophyll content of nutrient barley, and treatment of strain Bacillus cereus AST-4 increases the chlorophyll content of nutrient barley to help the growth of nutrient barley. Chlorophyll plays a role in making nutrients on its own by allowing plants to photosynthesize. That is, an increase in the content of chlorophyll helps the growth of plants.

This strain is Bacillus 16S rRNA of a celebrity in-house (Bacillus cereus) AST-4 (KCTC18752P) is GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTNGAAGCAACGCGAAGAACCTTACC AGGTCTTGACATCCTNTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATNTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATNTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGACAA can be.

The present invention can provide a microbial agent for promoting plant growth in a salt stress environment including the Bacillus cereus AST-4, a culture thereof, or a mixture thereof.

Plant growth promotion in the salt stress environment means that when the strain of the present invention is treated with a plant, the content of chlorophyll increases, and the photosynthesis of the plant increases with the increased content of chlorophyll, thereby increasing the nutrients in the plant. In a salt stress environment, plant growth may mean to increase plant photosynthesis in a salt stress environment or to increase chlorophyll in a plant in a salt stress environment.

The microbial preparation according to the present invention can be prepared in the form of a liquid fertilizer, and a bulking agent can be added thereto to be used in the form of a powdery powder or to be granulated by formulating it. However, the formulation is not particularly limited. In other words, it is possible to formulate bio-fertilizer to overcome this in eco-friendly organic farming where chemical fertilizer supply is limited.

In the salt stress environment, the salt concentration of the soil may be 3% to 10%, preferably 3% to 7%, and more preferably 5%.

The plant may be pear, apple, pepper, cabbage, radish, tomato, cucumber, watermelon, melon, carrot, sweet potato, cucumber, barley, wheat, corn and soybean, but is not limited thereto. Preferably it can be barley. In some cases, the growth of plants by microorganisms is not limited to specific plant species due to indirect action such as phosphoric acid solubilization, but may also be an interaction between specific microorganisms and specific plants by specific signaling substances. This means that even if it is known as Bacillus cereus of the same species, the potential for promoting the growth of barley in the salt soil proposed by the present invention is not great. Therefore, the growth efficiency of barley under the salt soil environment of strain AST-4 can be differentiated.

The present invention can provide a microorganism fertilizer for promoting plant growth in a salt stress environment, including the Bacillus cereus AST-4, a culture thereof, or a mixture thereof.

The microbial fertilizer can be irrigated to the foliage or plant roots of the plant when the plant is growing. In the case of the culture solution, it is appropriately diluted and used, from planting to planting in the packaging for sandpaper or irrigation. Spraying and irrigation recovery may vary depending on the condition of the plant, but it is possible from time to time every 3 or 5 days. The microbial fertilizer may be prepared in the form of a liquid, granular, or hydrating agent.

The present invention provides a method for promoting plant growth comprising the step of treating the microbial fertilizer to a plant or a plant seed.

The treatment method may be used by irrigating the microbial fertilizer in a liquid state, immersing or spraying the seeds of crops or coating the seeds, but is not limited thereto.

The present invention can provide a soil conditioner comprising Bacillus cereus AST-4, a culture thereof, or a mixture thereof.

The present invention can provide a method of improving soil in a reclamation area, a sea landfill, a vinyl house, or a salt collection site by using the soil improving agent.

The soil may have a salt concentration of 3% to 10% of the soil, preferably 3% to 7%, and more preferably 5% of the soil.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Separation of neobacteria

In order to separate the strains, crop root soil was collected from the soil of the crop cultivation facility. Soil 1 g was inoculated in 100 mL of TSB (tryptic soy broth) liquid medium to which NaCl 3% was added, and cultured by shaking at 30 ° C. for 4-5 days. After transferring 3 to 4 times to the medium of the same conditions, a single colony was selected by diluting smear on TSB solid medium to isolate the strain.

Example 2. Identification of new bacteria

The isolated strain was cultured in the same medium to extract genomic DNA. PCR reaction using primers 27F (AGA GTT TGA TCM TGG CTC AG: SEQ ID NO: 2) and 1492R (GGT TAC CTT GTT ACG ACT T: SEQ ID NO: 3), 16S rRNA gene (SEQ ID NO: 1) from the extracted genome

GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTNGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTNTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATG Amplification and purification the TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATNTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATNTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGACAA) to analyze the nucleotide sequence. The analyzed sequencing was sorted in the SILVA database (v132), and a phylogenetic tree was prepared using the ARB program and phylogenetic analysis was performed. As a result of identifying the new strain, it was confirmed that the new strain AST-4 belongs to Bacillus cereus (FIG. 1).

Example 3. Effect of promoting growth of plants of the new strain

Nutritious barley seeds were soaked in distilled water at 4 ° C. for 24 hours in a dark place, and the seeds were germinated for 3 days in a soil containing no NaCl. The inoculum was incubated with R2A medium containing 3% NaCl and incubated for 48 minutes at 28 ° C., and centrifuged at 5000 rpm for 10 minutes to suspend the cell pellet in 10 mL of PBS buffer for another 10 minutes at 5000 rpm. The supernatant was discarded by centrifugation and suspended in 5 mL of PBS buffer. The inoculum thus prepared was prepared by adjusting the absorbance to 1.0 at 600 nm with a spectrophotometer. In order to confirm the ability of the strain AST-4 to promote plant growth, 5 seed germinated seeds were transferred to each of 5 g of top soil per pot, and then treated with NaCl at a concentration of 40 g NaCl / kg soil (4%) and a total of 15 During the day, cultivation was performed at 22 ° C for 8 hours under dark conditions and 16 hours under dark conditions. The inoculum was inoculated with 5 mL of seeds on the 3rd and 10th day, respectively. After 15 days of cultivation, the stem length of nutrient barley and the chlorophyll concentration of leaves were measured. To extract the chlorophyll of the leaves, 100 mg of cultivated barley leaves were measured and put into 6 mL of 90% ethanol and heated to 80 ° C. After boiling for 5 minutes, it was cooled at room temperature for 10 minutes and stirred for 20 minutes. After 20 minutes, the supernatant was obtained by centrifugation at 3000 rpm for 10 minutes, and the absorbance was measured at 663 nm and 645 nm with a spectrophotometer to calculate chlorophyll concentrations using the following formulas (1), (2) and (3).

Chlorophyll a = 12.7 A663-2.69 A645 ......... (1)

Chlorophyll b = 22.9 A645-4.68 A663 ......... (2)

Total Chlorophyll (a + b) = 20.29 A645 + 8.02 A663 ......... (3)

After 15 days of total cultivation, barley showed the largest growth in the soil without 4% NaCl added, and 4% NaCl added, but showed the lowest growth in the soil not inoculated with strain AST-4, and the strain in 4% NaCl soil Barley growth was better in soils inoculated with AST-4 than in soils inoculated with strains. This was also observed in the chlorophyll content. Both chlorophyll a and b did not reach the soil without the addition of NaCl, but the content was higher in the soil inoculated with the strain AST-4 than in the soil not inoculated with the strain (FIG. 2).

Accession number: KCTC18752P

Depository Organization: Korea Research Institute of Bioscience and Biotechnology Biological Resource Center

Deposit date: 12 December 4, 2018

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> AST-4 promoting plant growth under salt condition and uses          thereof <130> 1064958 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1392 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA of AST-4 <400> 1 ttagcggcgg acgggtgagt aacacgtggg taacctgccc ataagactgg gataactccg 60 ggaaaccggg gctaataccg gataacattt tgaaccgcat ggttcgaaat tgaaaggcgg 120 cttcggctgt cacttatgga tggacccgcg tcgcattagc tagttggtga ggtaacggct 180 caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 240 acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 300 acggagcaac gccgcgtgag tgatgaaggc tttcgggtcg taaaactctg ttgttaggga 360 agaacaagtg ctagttgaat aagctggcac cttgacggta cctaaccaga aagccacggc 420 taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg 480 gcgtaaagcg cgcgcaggtg gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg 540 gagggtcatt ggaaactggg agacttgagt gcagaagagg aaagtggaat tccatgtgta 600 gcggtgaaat gcgtagagat atggaggaac accagtggcg aaggcgactt tctggtctgt 660 aactgacact gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 720 cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgaagttaac 780 gcattaagca ctccgcctgg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg 840 ggggcccgca caagcggtgg agcatgtggt ttaattngaa gcaacgcgaa gaaccttacc 900 aggtcttgac atcctntgac aaccctagag atagggcttc tccttcggga gcagagtgac 960 aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020 gcgcaaccct tgatnttagt tgccatcatt tagttgggca ctctaaggtg actgccggtg 1080 acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1140 acacgtgcta caatggacgg tacaaagagc tgcaagaccg cgaggtggag ctaatntcat 1200 aaaaccgttc tcagttcgga ttgtaggctg caactcgcct acatgaagct ggaatcgcta 1260 gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320 cacaccacga gagtttgtaa cacccgaagt tcggtggggt aaccttttgg agccagccgc 1380 ctaaggtgac aa 1392 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 3 ggttaccttg ttacgactt 19

Claims (6)

Bacillus cereus AST-4 (KCTC18752P), which has salt resistance at a salinity of 4% NaCl, increases the content of chlorophyll, and promotes the growth of barley. A microorganism preparation for promoting the growth of barley in a stress environment with a salinity of 4% NaCl, comprising the strain of claim 1, a culture thereof, or a mixture thereof. A microorganism fertilizer for promoting the growth of barley in a stressful environment with a salinity of 4% NaCl, comprising the strain of claim 1, a culture thereof or a mixture thereof. A method for promoting the growth of barley in a stressful environment with a salinity of 4% NaCl comprising the step of treating the microbial fertilizer of claim 3 to barley or barley seeds. delete delete

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