KR100646601B1 - 083 91050 Bacillus sp. KR083KACC91050 promoting the growth of crops and the microbial agent containing the same - Google Patents

Abstract

The present invention relates to Bacillus sp KR083 (KACC91050) of SEQ ID NO: 1, which is a microbial strain that helps plant growth in place of chemical fertilizers. In more detail, the present invention is a microbial strain Bacillus sp. KR083 (KACC91050) and microorganisms, which are microorganism strains that settle on and inside the root surface of plants to fix aerial nitrogen, promote plant growth, and inhibit the growth of plant pathogens. It relates to a formulation.

Bacillus sp KR083 (KACC91050) * Freeze nitrogen * Promote plant growth * Microbial fertilizer

Description

Microorganism Bacillus sp. VR083 (BACC91050) for promoting crop growth and microbial preparations containing the same {Bacillus sp. KR083 (KACC91050) promoting the growth of crops and the microbial agent containing the same}

Figure 1 shows the separation operation of Bacillus sp KR083 (KACC91050).

2A and 2B are electron micrographs of Bacillus sp. KR083 (KACC91050).

3 is a photograph of the Bacillus sp KR083 (KACC91050) is settled inside the plant roots.

4 is a microbial deposit of Bacillus sp KR083 (KACC91050).

The present invention relates to Bacillus sp. KR083 (KACC91050) of SEQ ID NO: 1, which is a microbial strain that helps plant growth of plants instead of chemical fertilizers. In more detail, the present invention is a microbial strain Bacillus sp KR083 (KACC91050) and comprises the same to settle on the inside and inside the root of the plant to fix the nitrogen and promote the growth of plants, inhibiting the growth of plant pathogens It relates to a microbial agent.

In agriculture, fertilizers are important agricultural materials that determine the yield of crops, and if they are used too much, they cause eutrophication of river water, water pollution of groundwater, imbalance of soil nutrients, and poor growth of crops. It can cause the quality of However, the use of fertilizer to increase the productivity of crops is inevitable due to the reduction of agricultural land area due to the development and diversification of the industry. Accordingly, there is increasing interest in the development of environmentally friendly functional agricultural materials than conventional chemical fertilizers.

On the other hand, cultivation of crops using soil microorganisms is widely done in legumes, and the microorganisms used at this time are Rhizobium (Rhizobium) which has a symbiotic relationship with legumes. Among the soil microorganisms, microorganisms that promote plant growth without having a symbiotic relationship with host plants were named Plant Growth-promoting rhizobacteria (PGPR) by Kloepper in 1980. Representative microorganisms include Azospirrilum (Okon, 1985), Pseudomonas (Kloepper et al., 1980), and Bacillus (Bacillus, Backman, 1994). These plant growth-promoting microorganisms include fixed nitrogen (Boddy and Dobereiner, 1995), production of plant growth hormones, promotion of plant nutrient absorption (Hallmann et al., 1997), and plant disease inhibition (Pleban et al., 1995). It is known to help plant growth by various functions. The plant inoculation effects of various plant growth-promoting microorganisms that help plant growth have been increased from 19 to 60% (Baldani, 2000) in rice, tomatoes (Kloepper and Schroth, 1981), soybeans (Polonenko et al., 1987; Dashti, etc.). , 1998), wheat (Freitas and Germida, 1990), sugar beets (Suslow and Schrith, 1982), and the like, and research on the practical use of these plant growth-promoting microorganisms is ongoing.

In addition, Ward et al. (1992) said that less than 5% of all species of microorganisms in the soil were classified and identified, and Young (1992) said that more than 100 microorganisms were used to fix aerial nitrogen. 1990) reported that there are many bacteria in the soil that are not artificially cultured. Therefore, it is very likely that microbial strains that are functionally superior to useful microorganisms already found are found.

However, until now, among the soil microorganisms classified as the rhizosphere microorganisms that promote plant growth, one species of a specific microorganism has a function that simultaneously performs the function of fixation of aerial nitrogen, secretion of plant growth-promoting hormone, and inhibition of plant disease. Little is known about it.

In this regard, the inventors of the present invention have suggested that plant rootnote bacterium Bacillus sp. KR083 (KACC91050), which has the characteristics of settling on and inside of rice roots, is a single species of aerial nitrogen fixation, plant growth-promoting hormone secretion, and The present invention was completed by discovering that it has a function such as suppressing disease occurrence.                         

Accordingly, it is an object of the present invention to provide a Bacillus sp. KR083 (KACC91050) strain having functions such as nitrogen fixation, plant growth-promoting hormone secretion, and inhibition of plant disease.

It is also an object of the present invention to provide a secondary metabolite of the Bacillus sp. KR083 (KACC91050) strain.

In addition, another object of the present invention is to provide a microbial agent comprising the Bacillus sp KR083 (KACC91050) strain as an active ingredient.

The present invention is a microbial strain SEQ ID NO: 1 Bacillus sp. KR083 (KACC91050) and a microbial preparation comprising the same microorganism strain to settle on the surface and inside the root of the plant to fix the nitrogen and promote the growth of the plant, inhibiting the growth of plant pathogens It is about.

Hereinafter, the present invention will be described in detail.

KR083 has a round bar shape at both ends and has a size of 1.5-2.0 × 0.8-1.0 μm with flagella (FIG. 2). Poly-beta-oxybutyrate accumulates when Bacillus sp. KR083 (KACC91050) strain is cultured in PDB medium for 24 hours. Gram-positive bacteria, KR083, have spores, respiratory metabolism, and catalase activity, depending on the growth environment. In addition, the KR083 strain is capable of fixing molecular nitrogen (N ₂ ) and growing in 7% sodium chloride (NaCl) solution, and is colorless in litmus milk reaction. The KR083 strain produces acid during growth when the carbon source added to the medium is Glucose, Arabinose, Xylose, Galactose, Fructose, Lactose, Sucrose, Mannitol, etc. Produces alkali On the other hand, Dulcitol and Glycerol supplemented medium did not grow. In addition, the KR083 strain has a property of entering into the root tissue while inhabiting the surface of the plant root (FIG. 3). Physiological and biochemical characteristics of the soil microbial KR083 strain are shown in Table 1 below.

Physiological and Biochemical Characteristics of Soil Microbial KR083 Strains division Main characteristic division Main characteristic Strain shape Rod shape Strain size (μm) 1.5-2.0 × 0.8-1.0 flagellum has exist      Create poly-beta-oxybutyrate Produced after incubation in PDB medium Spores has exist Gram Dyeing + Aerobic metabolism + Anaerobic metabolism - Nitrogen fixation + Catalase Activity + Litmus milk reaction Colorless reaction Denitrification + Starch hydrolysis - Casein hydrolysis - Lipase Activity - Growth at 4 ℃ + 7% sodium chloride (NaCl) solution growth + Carbon source reaction                                                                                                                                                                                                                                                                                                                              Glucose Acid production Carbon source reaction                                                                                                                                                                                                                                                                                                                              Maltose Acid production Arabinose Acid production Mannitol Acid production Xylose Acid production Sorbitol Alkaline Generation Lactose Acid production Ducitol Inability to grow Galactose Acid production Glycerol Inability to grow Fructose Acid production Dextrin Alkaline Generation Ramnose Alkaline Generation Starch Alkaline Generation Sucrose Acid production Citrate Alkaline Generation

Reference Example 1 Sequence Analysis of the KR083 Strain

The nucleotide sequence analysis of the KR083 strain was performed by 16S rDNA sequence analysis. First, the 16S rDNA region was amplified by a nucleic acid amplification method (PCR: Polymerase Chain Reaction), inserted into a pGEMT-T vector, and then the automatic sequencer ABI3100 ( Applied Biosystem, USA) was analyzed for sequencing. The primers used here were fD1 (5'-agagtttgatggctcag-3 ') of SEQ ID NO: 2 and rP2 (5'-acggctaccttgttacgactt-3') of SEQ ID NO: 3, and analyzed the nucleotide sequence of the sock end of the primer. For this purpose, a forward primer (5'-gttttccca gtcacgac-3 ') (SEQ ID NO: 4) and a reverse primer (5'-gcggataacaatttcacacagg-3') (SEQ ID NO: 5) were used. In addition, four primers were used for internal sequencing. Each base sequence is 16SP-1 (5'-gccacactggaactgagacac-3 '), which is the base sequence of 311-330 sites, and the 684-703 site sequence, according to the sequence of Escherichia coli 16S rDNA (GenBank No. J01695). 16SP-2 (5'-tgtagcg gtgaaatgcgtg-3 '), 16SP-3 (5'-ggagcatgtgt tattcg-3) which is the nucleotide sequence of 944-963 site, and 16SP-4 (5'- which is the nucleotide sequence of 1228-1247 site, ctacacacgtg ctacaatgg-3 ').

The 16S rDNA sequencing results of the KR083 strain are shown in SEQ ID NO: 1. As a result of investigating the homology with the nucleotide sequence of the GenBank library, it showed high nucleotide sequence homology of over 99% with Bacillus sp. It was classified as Bacillus sp. Because it showed relatively low sequence homology of less than 98%. Accordingly, the present inventors named the KR083 strain as the strain of Bacillus sp. KR083 and deposited it at the Korea Agricultural Microbiological Conservation Center (KACC), attached to the Rural Development Administration, on June 11, 2003, and received the accession number KACC91050.

Plant growth promoting power of Bacillus sp KR083 (KACC91050) strain according to the present invention was found that when the roots were inoculated in a 50-fold dilution of the strain culture by 1.5 hours, the root height was increased by 21.3%, aerial nitrogen The fixation force was 1.02 times and 143 times higher than the standard strains Herbaspirrilum seropedicae BR11175 and Acetobacter diazotrophicus BR11281, respectively, at 72 hours at room temperature. It showed strong growth inhibitory effect against two kinds of bacteria including Erwinia carotovora.

The microbial preparation according to the present invention can be formulated as a preparation for promoting plant growth or inhibiting pathogen growth in a conventional manner, and is not particularly limited to the formulation. Preferably, it may be formulated as a plant growth promoting organic fertilizer to replace the chemical fertilizer.

In addition to the Bacillus sp KR083 (KACC91050) per g of the dry powder isolates, or liquid fertilizer at least 10 ⁵ per 1㎖ if the production of the microorganism preparation according to the present invention in dry powder form or in the form of a liquid fertilizer formulations for plant growth promotion It may contain.

In addition, when the microbial preparation according to the present invention is prepared in the form of a dry powder or liquid form as a preparation for inhibiting pathogen growth, the Bacillus sp KR083 (KACC91050) strain may contain 10 ⁷ or more per gram of dry powder or 1 ml of liquid. It can be, characterized in that the use of more content of the strain than when formulated into the agent for promoting plant growth.

Hereinafter, although an Example and an experimental example are given and this invention is demonstrated further more concretely, this invention is not necessarily limited to these Examples and an experimental example.

Example 1 Isolation of Soil Microorganism KR083

Aseptically germinated rice seed grains were transferred onto 0.6% agarose medium, inoculated with 0.5 g of soil sample to isolate the strain at 1-2 cm distance from rice roots, and incubated at 28 ° C. for 48 hours. Grind was made into a suspension, and then plated on an LC medium to isolate strains. The isolated KR083 strain is shown in FIG. 1.

Experimental Example 1 Aerial Nitrogen Fixation of Soil Microorganism KR083

The KR083 strain was inoculated in 5 ml of semi-solid LGI medium in a vacuum vessel, and then incubated and assayed by measuring acetylene reducing power (Hardy, 1970) by gas chromatography (Gas Chromatography). The results are shown in Table 2 below.

Aerial Nitrogen Fixation of Soil Microorganism KR083 Strain Strain activity by incubation time (umol C ₂ H ₄ /24mL/0.5hr.) 12hr 24hr 48hr Herbaspirillum seropedicae BR11175 0.93 7.89 170.93 Herbaspirillum rubrisublbican BR11192 0.64 0.83 3.70 Acetobacter diazotrophicus BR11281 0.45 0.81 1.29 KR083 0.45 21.48 249.68

As shown in Table 2, KR083 strain of the present invention showed a better public nitrogen fixation than the control strain Herbaspirillum seropedicae BR11175, Herbaspirillum rubrisublbican BR11192 and Acetobacter diazotrophicus BR11281.

Experimental Example 2 Plant Growth Promoting Effect of Soil Microorganism KR083

In order to measure the plant growth promoting effect of the KR083 strain, we tested the alga Chlorella vulgaris and corn seedlings. Chlorella vulgaris inoculation test is when the temperature of Hata medium is 50 ℃, mixed Chlorella vulgaris in a liquid medium and poured into a curry to harden the medium, and then make a groove of 4 mm radius to make 100 µl of the strain culture solution in 10 repetitions. Busy. Treated algae were incubated under light conditions for 5 days, and then grown. Corn seedling inoculation test was inoculated by soaking the root portion of the corn root 3 mm in the culture suspension of the test strain for 1.5 hours, the seedlings of the control was immersed in sterile microbial culture. The corn seedlings were then moistened in a wet filter paper in a plastic box, covered with a root tip at the beginning of the kidney extension, and incubated at 28 ° C. for 24 hours to measure the length of the newly extended roots 10 times and then to measure the corn. Growth promoter was assayed. The results are shown in Table 3 below.

Soil microorganism KR083 Monarch algae and corn growth promoter Strain Algal Growth (Φ, mm / 5 days)        Corn growth (% of untreated) 1: 1 1:50 KR083 3 -20 +26.3 Control strain Chryseobacterium sp. KR044 0 0 +8 Control strain Bacillus sp. KR091 0 0 0

As shown in Table 3, KR083 strain showed an effect of promoting the growth of algae 3mm in diameter on the 5th day of inoculation, in the case of measuring the corn root height, twice the culture stock (4.5 × 10 ⁹ cfu / ㎖) Diluted inoculation showed 20% root elongation inhibition effect compared to the untreated group. However, 50-fold dilution of the culture showed 26.3% elongation promoting effect compared to the untreated group.

Experimental Example 3 Inhibitory Activity of Plant Pathogens of Soil Microorganism KR083

Inhibition of phytopathogenic bacteria of KR083 strain was inoculated with pathogenic fungi and bacteria in PDA (Potato Dextrose Agar) and transferred to a shard to harden the medium, and then made a well of a diameter of 4 mm, which was 28 ° C. in a liquid potato medium. After inoculating 100 μl of KR083 strain cultured for 5 days under conditions, stored at 28 ° C. for 5 days, the extent of growth inhibition of pathogens of rhizosphere bacteria was measured. Inoculation of phytopathogens was performed by mixing 5 ml at 1 × 10 ⁶ conidia / ml concentration for fungi and 5 ml at 1 × 10 ⁸ cfu / ml concentration for bacterial pathogens. Inoculated by repeating three aliquots 25ml each. The results are shown in Table 4 below.

Growth Inhibition of Phytopathogenic Fungi and Bacteria of Soil Microbial KR083 Strains (Growth Range, Φ㎜) Strain        Phytopathogenic fungi Phytopathogenic Bacteria Rhizoctonia solani Fusarium culmorum Erwinia carotovora 3391 Pseudomonas syringiae 8300 KR083 30.7 ± 1.7 34.0 ± 7.0 32.0 ± 3.2 26.0 ± 4.6

As shown in Table 4, the KR083 strain includes phytopathogenic fungi such as Rhizoctonia solani and Pythium, and Erwinia carotovora and Pseudomonas syringiae. It has been confirmed to exhibit a strong growth inhibitory effect on phytopathogenic bacteria such as.

Experimental Example 4 Inoculation Effects of Crop Crops by Soil Microorganism KR083

The plant growth promoting soil microorganism Bacillus sp KR083 (KACC91050) strain of the present invention was inoculated with barley, wheat, cabbage, sorghum and the like. The results are shown in Table 5 below.

Crop Inoculation Effects of Soil Microbial KR083 Strains Water  Quantity (kg / 10a) % Increase No vaccination inoculation   Barley (large barley 1) 733.5 762.0 3.9   Chili Pepper 376.8 428.5 13.7   Tomato (sun tomato, live weight 40 days) 1.66 g 4.41 g 165.7

As shown in Table 5, by inoculating Bacillus sp KR083 (KACC91050) strain barley yield was increased by 3.9%, pepper yield was 13.7%, tomato seedling growth was improved by 165.7%, respectively, according to these results KRO83 strain actually It was confirmed that the yield of the crop was increased.

As described above, Bacillus sp KR083 (KACC91050) of the present invention is fixed to the root surface and inside of the plant to fix the aerial nitrogen, promote the growth of the plant, and confirmed that it is effective in inhibiting the growth of plant pathogens It became. Therefore, when Bacillus sp KR083 (KACC91050) is supplied as a microbial agent by a conventional method, it can not only preserve the water quality and the soil environment, but also increase the crop yield by replacing chemical fertilizers, Can help to grow safely.

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Claims (7)

Bacillus sp. KR083 (KACC91050) strain having the rDNA nucleotide sequence of SEQ ID NO: 1. A microbial preparation comprising Bacillus sp KR083 (KACC91050) according to claim 1 as an active ingredient. According to claim 2, wherein the microbial agent is a microbial agent, characterized in that the fertilizer replacement organic fertilizer. The microorganism preparation according to claim 2, wherein the microbial preparation is for promoting plant growth. The microbial preparation of claim 2, wherein the microbial preparation is for inhibiting pathogen growth. The microbial preparation of claim 2, wherein the microbial preparation further contains a secondary metabolite of Bacillus sp KR083 (KACC91050) as an active ingredient. A soil management and plant cultivation method for treating the microorganism preparation according to any one of claims 2 to 6 to soil or plants.

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