KR20050116297A - Pseudomonas sp. rrj228 helping the growth of crops and the microbial agent containing the same - Google Patents

Abstract

The present invention relates to the Pseudomonas sp. RRj228 (KACC91052) strain of SEQ ID NO: 1, which promotes the growth of plants by fixing on the inside and inside of the roots of plants instead of chemical fertilizers during crop cultivation.

The present invention also relates to an environment-friendly microbial agent containing the Pseudomonas sp. RRj228 (KACC91052) strain and the secondary metabolite produced by the strain.

Environmentally-friendly microbial preparations according to the present invention can improve the fertility reduction, stabilization of crop productivity, conservation of water quality and soil environment, and soil ecosystem by using the Pseudomonas sp. RRj228 (KACC91052) strain.

Description

Pseudomonas sp. R.228, which aids in the growth of crops, and microbial preparations containing the same {Pseudomonas sp. RRj228 helping the growth of crops and the microbial agent containing the same}

The present invention relates to the Pseudomonas sp. RRj228 (KACC91052) strain of SEQ ID NO: 1, which promotes the growth of plants by fixing on the inside and inside of the roots of plants instead of chemical fertilizers during crop cultivation.

The present invention also relates to an environment-friendly microbial agent containing the Pseudomonas sp. RRj228 (KACC91052) strain and the secondary metabolite produced by the strain.

In agriculture, fertilizers are important agricultural materials that determine the yield of crops, but if they are used excessively, they cause eutrophication of river water, water pollution of groundwater, imbalance of soil nutrients, and poor quality of crop growth, resulting in a decrease in yield and quality of agricultural products. . Therefore, proper utilization of soil microorganisms, which make plant growth strong while partially replacing existing chemical fertilizers, was necessary for the development of sustainable agriculture and the preservation of the agricultural environment.

Microorganisms that promote the growth of standing plants do not have a complete symbiosis with the host plant from the soil microorganism was named as PGPR (Plant growth-promoting rhizobacteria) such as by Kloepper 1980 years, here it is Azospirrilum spp securing the air of nitrogen (Okon , 1985), Pseudomonas genus (Kloepper et al., 1980) and Bacillus genus (Backman et al., 1994). Plant growth-promoting microorganisms, called PGPR strains, include the fixation of aerial nitrogen (Boddy and Dobereiner, 1995), the production of plant growth hormones, the promotion of plant nutrient absorption (Hallmann et al., 1997), and the suppression of plant disease (Pleban et al., 1995). It is known to help plant growth by various functions such as).

Ward et al. (1992) reported that less than 5% of all species were identified and identified among soil microorganisms. Torisvik et al. (1990) found that many soils were not artificially cultured in the soil. Therefore, there will be another strain that is more functional than the useful microorganisms already discovered, and research on this continues.

In addition, the crop inoculation effects of various plant growth-promoting microorganisms to help plant growth were increased from 19 to 60% (Baldani, 2000) in rice, tomatoes (Kloepper and Schroth, 1981), beans (Polonenko et al., 1987; Dashti, etc.). , 1998), wheat (Freitas and Germida, 1990), sugar beet (Suslow and Schrith, 1982), and research on the practical use of these microorganisms is being promoted.

Therefore, the present inventors, while studying the soil microorganisms inhabiting the soil, was the first to isolate and identify the plant root wound bacteria Pseudomonas sp. RRj228 (KACC91052) strains that have the characteristics of settling on and inside the rice roots, this strain is the crop The present invention has been completed by discovering that growth can be substituted for conventional fertilizer.

Accordingly, it is an object of the present invention to provide a Pseudomonas sp. RRj228 (KACC91052) strain having a partial growth function of chemical fertilizer with the ability to promote plant growth to help crop growth.

It is also an object of the present invention to provide a microbial agent containing the Pseudomonas sp. RRj228 (KACC91052) strain and the secondary metabolite produced by the strain as an active ingredient.

The present invention relates to a Pseudomonas sp. RRj228 (KACC91052) strain of SEQ ID NO: 1, which is settled on and inside the root surface of a plant to promote plant growth.

Pseudomonas sp. RRj228 (KACC91052) strain according to the present invention is the first soil bacteria discovered by the present inventors, settles on the root surface and inside of the crop under natural conditions to secrete plant growth promoting substances, and against phytopathogenic fungi and bacteria It has antagonistic ability to inhibit the growth of various phytopathogens.

Pseudomonas sp. RRj228 (KACC91052) strain according to the present invention is a gram-negative bacteria in the shape of a stick and is a mesophilic bacterium that grows well in PDA (Potato Dextrose Agar) medium at 28 ± 2 ° C.

The effects obtained by using the Pseudomonas sp. RRj228 (KACC91052) strain of the present invention are as follows:

1) Pseudomonas sp. RRj228 (KACC91052) strain of the present invention is excellent in the nutrient supply power and plant growth promoting power of the plant can be used to produce a microbial preparation in a conventional way as a crop inoculation agent can increase the yield with less chemical fertilizer.

2) Pseudomonas sp. RRj228 (KACC91052) strain of the present invention has a wide application range of crops, such as rice, pepper, cucumber as an alternative fertilizer.

3) Pseudomonas sp. RRj228 (KACC91052) strain of the present invention has the property of inhibiting the growth of phytopathogenic microorganisms in the soil to help the safe growth of crops from pathogens when inoculated into crops.

4) Pseudomonas sp. RRj228 (KACC91052) strain of the present invention has a property of settling inside the root tissue of the crop has a high stability of crop inoculation effect expression.

5) Pseudomonas sp. RRj228 (KACC91052) strain of the present invention by separating and purifying the secondary metabolite from the culture in a conventional manner to create a plant value of promoting growth or plant pathogen growth inhibitory by pure separation to create value added over the chemical fertilizer replacement effect can do.

6) Since Pseudomonas sp. RRj228 (KACC91052) strain of the present invention is a microbial resource with no potential for environmental pollution in agriculture, when used for crop cultivation can be expected to preserve the water quality and soil environment and enhance the health of the soil ecosystem.

The present invention also relates to an environment-friendly microbial agent containing the Pseudomonas sp. RRj228 (KACC91052) strain and the secondary metabolite produced by the strain.

The microbial preparation according to the present invention may 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, and preferably as a plant growth promoting organic fertilizer for replacing a chemical fertilizer. It can be formulated.

In addition, 10 ⁵ to 10 ⁷ per 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 is the Pseudomonas sp RRj228 (KACC91052) or liquid fertilizer per the dry powder strain g 1㎖ It can be contained as horses.

In addition, when the microbial preparation according to the present invention is prepared in the form of dry powder or liquid form as a preparation for inhibiting pathogen growth, the strain Pseudomonas sp. RRj228 (KACC91052) 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 a higher content of the strain than when formulated with the agent for promoting plant growth.

When the Pseudomonas sp. RRj228 (KACC91052) strain according to the present invention is properly used as a substitute for chemical fertilizers, chemical fertilizer reduction, stabilization of crop productivity, conservation of water quality and soil environment, and improvement of soil ecosystem health can be expected.

Pseudomonas sp. RRj228 (KACC91052) strain can be prepared in the form of a suitable microbial preparation in general cropland soil, and can be inoculated into crops such as rice, red pepper, and cucumber instead of chemical fertilizer.

Isolation, identification and plant growth of Pseudomonas sp. RRj228 (KACC91052) strain of the present invention excellent in plant growth promoting ability isolated from Russia St. Petersburg among 172 strains isolated from the soil of Korea and Russia according to the following examples Explain acceleration.

However, the following examples are only examples given to practice the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Isolation of Soil Microbial RRj228 Strain

Separation of RRj228 strains of plant root zone soil microorganisms was inoculated with 0.5 g of a soil sample to isolate the strain 1 to 2 cm away from rice roots by transferring one sterile germinated rice seed as shown in Fig. 1 onto 0.6% agarose medium. After 48 hours of incubation at 28 ° C, the roots were ground to form a suspension, followed by Elci (LC) medium (10 g of bactotrypton, 5 g of yeast extract, 8 g of MgSO ₄ · 7H ₂ O, 1 M Tris 1 ml, distilled water 1 L, pH 6.8) was plated to isolate the strain.

Example 2 Identification of Soil Microorganism RRj228

end. Physiological and biochemical properties

Physiological and biochemical characteristics of the RRj228 strain are shown in Table 1 below.

The RRj228 strain had a rod shape, a size of 2.0-3.0 × 0.5-0.7 μm, and had flagella as shown in FIGS. 2A and 2B. In PDB medium, colony color was reddish yellow after 24 hours of incubation. Gram negative bacterium, RRj228, is respiratory metabolism and has catalase and oxidase activity. The Pseudomonas sp. RRj228 strain contains arabinose, fructose, galactose, glucose, mannitol, sucrose, and xylose. ) Produced acid during growth, and alkaline when carbon source was lactose, dextrin, ramnose, sorbitol, maltose, maltose and dulcitol. . In addition, the RRj228 strain has a property of entering into the root tissue and settling while inhabiting the plant root surface as shown in FIGS. 3 and 4.

TABLE 1

Physiological and Biochemical Characteristics of Soil Microbial RRj228 Strains

division Main characteristic division Main characteristic Strain shape Rod shape Strain size (μm) 2.0-3.0 X 0.5-0.7 flagellum has exist Spores none Gram Dyeing - Colony color on PDDA badge Red yellow Aerobic metabolism + Anaerobic metabolism - Catalase Activity + Oxidase Activity + Gelatin hydrolytic power + Lipase Activity - Starch hydrolysis - Casein hydrolysis + Carbon source reaction Glucose Acid generation Carbon source reaction Sucrose Acid generation Arabinose Acid generation maltose Alkaline Generation Xylose Acid generation Mannitol Acid generation Lactose Alkaline Generation Sorbitol Alkaline Generation Galactose Acid generation Ducitol Alkaline Generation Fructose Acid generation Glycerol Acid generation Rhamnose Alkaline Generation dextrin Alkaline Generation

I. Sequencing

The sequencing of the RRj228 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 base sequencer ABI3100 (Applied Biosystem, USA) to analyze the base sequence. 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 the pUC / M13 forward primer (5'-gttttccca gtcacgac-3 ') (SEQ ID NO: 4) and reverse primer (reverse primer: 5'-gcggataacaatttcacacagg-3') (SEQ ID NO: 5) were used. Four primers were used for analysis.

Each nucleotide sequence is 16SP-1 (5'-gccacactggaactgagacac-3 '), which is a nucleotide sequence of 311-330 site, according to the sequence of Escherichia coli 16S rDNA nucleotide sequence (GenBank No. J01695), which is a nucleotide sequence of 684-703 site. 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 ').

16S rDNA sequence of the RRj228 strain is shown in SEQ ID NO: 1, and the homology with the genome of GenBank library showed that the sequence homology of 99% or more of Pseudomonas species such as Pseudomonas chlorororaphis, P. Corrugata, P. tolaasii It was classified as Pseudomonas sp.

Therefore, the present inventors named the RRj228 strain as "Pseudomonas sp. RRj228 strain", and deposited an accession number KACC 91052 on June 11, 2003, which was deposited with the Korea Agricultural Microbiological Conservation Center (KACC), which was established by the Rural Development Administration. .

[Test Example 1] Plant growth promoting effect assay of Pseudomonas sp. RRj228 strain

Plant growth promoting effect assay test of Pseudomonas sp. RRj228 strain was conducted on corn seedlings. 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 are then placed in a damp filter paper in a plastic box, covered with the end of the root at the starting line of the roots, and then incubated at 28 ° C. for 24 hours to lengthen the newly extended roots. Was repeated 10 times, and the results of assaying the corn growth promoter of this RRj228 strain are shown in Table 2 below.

TABLE 2

Corn Growth Enhancement of Pseudomonas sp. RRj228

Strain Corn growth (% of untreated) 1:50 1: 100 RRj228 -26 +13.0 Control strain Chryseobacterium sp. KR044 0           +8 Control strain Bacillus sp. KR091 0            0

As can be seen in Table 2, the plant growth promoting power of the Pseudomonas sp. RRj228 (KACC91052) strain according to the present invention when the roots were inoculated by immersing the corn roots in the strain culture solution for 1.5 hours (4.5 × 10 ⁹ cfu / ml) was reduced by 26% at 50-fold dilution, but increased by 13.0% at 100-fold dilution.

Test Example 2 Soil Disease Antagonist Assay of Pseudomonas Esp RRj228 Strain

Soil disease antagonism assay of Pseudomonas sp. RRj228 strain was inoculated with pathogenic fungi and bacteria in PDA and transferred to Shale to harden the medium, and then formed a well of 4 mm radius 100 μl of Pseudomonas sp. RRj228 strain incubated at 28 ° C. for 5 days in medium was stored at 28 ° C. for 5 days, and then the growth inhibition range of myobacterial bacteria was measured, and the results are shown in Table 3. Inoculation of phytopathogens was performed by mixing 1 × 10 ⁶ conidia / ml 5ml and 1 × 10 ⁸ cfu / ml 5ml of bacterial pathogens in 250ml of PDA medium at 45 ° C. Three ml aliquots were dispensed and inoculated.

TABLE 3

Growth Inhibition of Phytopathogenic Fungi and Bacteria of Soil Microbial RRj228 Strain ^* (Growth inhibition range, Φ㎜)

Strain        Phytopathogenic fungi Phytopathogenic Bacteria Phytophtora capsici      Pythium Erwinia carotovora 3391 Pseudomonas syringiae 8300 RRj228 49.3 ± 6.3 57.3 ± 6.3 56.0 ± 1.2 50.0 ± 4.6

^* Range of growth inhibition of pathogen 5 days after strain inoculation

As can be seen from the above results, RRj228 strain according to the present invention is Phytopathogenic fungus Phytophtora capsici (Pythium) and Pythium (Pythium), phytopathogenic bacteria Erwinia carotovora and Pseudomonas syringe Pseudomonas syringiae showed a strong growth inhibitory effect.

Test Example 3 Root engraftment competence of Pseudomonas sp. RRj228 strain

Competitiveness of plant root cultivation of test strain was RRj228 among all strains grown on root when Pseudomonas fluorescens WCS365 and control strain were inoculated in the roots of rice and Saxa nova. It is represented by the ratio occupied by the strain, the results are shown in Table 4.

TABLE 4

Root rot competence compared to Pseudomonas fluorescens WCS365 strain of soil microbial Pseudomonas sp. RRj228

Strain Root Growth Density (10 ⁵ cfu / cm. Root) Root Grow Share (%) Rice Saxa nova Rice Saxa nova Pseudomonas Esp RRj228 7.90 (1,975) 72.5 (145) 95.2 99.3 Pseudomonas Fluorescens WCS365 0.40 (100) 0.5 (100) 4.8 0.7

Test Example 4 Inoculation Effect of Pseudomonas Esp RRj228 Strains

The plant growth promoting soil microorganism Pseudomonas sp. RRj228 strain (KACC91052) of the present invention was inoculated in rice, pepper, cucumber, tomato cultured in a conventional manner, the results are shown in Table 5 below.

TABLE 5

Crop Inoculation Effects of Soil Microbial RRj228 Strains

Water  Quantity % Increase No vaccination inoculation   Rice (Dasan Rice, Pot cultivation for 3 weeks) 40.4 g 53.8 g 33.2   Red pepper (Cotton cultivated with 4 weeks of hot pepper) 376.8 g 408.7 g 8.5   Cucumber (Dinus Tio) (live weight 40 days) 4.25 g 5.16 g 21.4   Tomato (sun tomato) (live 40 days sowing) 1.66 g 3.61 g 117.5

As shown in Table 5, the Pseudomonas sp. RRj228 strain (KACC91052) improved rice yield by 33.2%, pepper yield by 8.5%, cucumber growth by 21.4%, and tomato growth by 117.5%. It was confirmed that Pseudomonas sp. RRj228 strain can be used as a microbial fertilizer resource that can replace a considerable amount of chemical fertilizer.

Pseudomonas sp. RRj228 (KACC91052) strain according to the present invention, since it is directly separated from the root of the plant (rice), unlike the conventional strain directly isolated from the soil, because the characteristics to be settled inside the crop roots of rice, cucumber, etc. When inoculated, the roots can grow on or inside the roots, and as long as the crop is alive, the effect of inoculation can be long because the density decreases due to rain or drying.

In addition, the environment-friendly microbial fertilizer composition according to the present invention by using the Pseudomonas sp. RRj228 (KACC91052) strain, can partially replace the chemical fertilizer when cultivating crops to increase the soundness of the agricultural environment, such as water quality, soil and improve crop productivity Can contribute to

1 shows plates cultured for isolation of Pseudomonas sp. RRj228 (KACC91052) strain.

2A and 2B are electron micrographs of Pseudomonas sp. RRj228 (KACC91052) strain.

Figure 3 is a photograph of the Pseudomonas sp. RRj228 (KACC91052) strain is settled inside the plant roots.

4 is a deposit of Pseudomonas sp. RRj228 (KACC91052) strain.

<110> Republic of Korea represented by Rural Development Administration <120> Pseudomonas sp. RRj228 promoting the growth of the farm produce and the manure composition containing the same <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1501 <212> DNA Pseudomonas sp. RRj228 <400> 1 gagtttgatc ctggctcaga ttgaacgctg gcggcaggcc taacacatgc aagtcgagcg 60 gtagagagaa gcttgcttct cttgagagcg gcggacgggt gagtaatgcc taggaatctg 120 cctggtagtg ggggataacg tccggaaacg gacgctaata ccgcatacgt cctacgggag 180 aaagcagggg accttcgggc cttgcgctat cagatgagcc taggtcggat tagctagttg 240 gtgaggtaat ggctcaccaa ggcgacgatc cgtaactggt ctgagaggat gatcagtcac 300 actggaactg agacacggtc cagactccta cgggaggcag cagtggggaa tattggacaa 360 tgggcgaaag cctgatccag ccatgccgcg tgtgtgaaga aggtcttcgg attgtaaagc 420 actttaagtt gggaggaagg gtacttacct aatacgtgag tattttgacg ttaccgacag 480 aataagcacc ggctaactct gtgccagcag ccgcggtaat acagagggtg caagcgttaa 540 tcggaattac tgggcgtaaa gcgcgcgtag gtggttcgtt aagttggatg tgaaatcccc 600 gggctcaacc tgggaactgc atccaaaact ggcgagctag agtatggtag agggtggtgg 660 aatttcctgt gtagcggtga aatgcgtaga tataggaagg aacaccagtg gcgaaggcga 720 ccacctggac atgatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgtc aactagccgt tgggagcctt gagctcttag 840 tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg ttaaaactca 900 aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggccttg acatccaatg aactttccag agatggattg gtgccttcgg 1020 gaacattgag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg ggcactctaa 1140 ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1200 acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa gccgcgaggt 1260 ggagctaatc ccataaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1320 agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc atgggagtgg gttgcaccag aagtagctag tctaaccttc 1440 gggaggacgg ttaccacggt gtgattcatg actggggtga agtcgtaaca aggtagccgt 1500 a 1501 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PCR amplification primer fD1 for amplifying the gene of Pseudomonas sp. RRj228 <400> 2 agagtttgat ggctcag 17 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR amplication primer rP2 for amplifyign the gene of Pseudomonas sp. RRj228 <400> 3 acggctacct tgttacgact t 21 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PCR amplification forward primer of pUC / M13 for amplifying the gene of Pseudomonas sp. RRj228 <400> 4 gttttcccag tcacgac 17 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR amplification reverse primer of pUC / M13 for amplifying the gene of Pseudomonas sp. RRj228 <400> 5 gcggataaca atttcacaca gg 22

Claims (7)

Pseudomonas sp. RRj228 (KACC91052) strain having the rDNA sequence of SEQ ID NO: 1. Microbial agent containing the Pseudomonas sp. RRj228 (KACC91052) strain 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 microbial preparation of 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 Pseudomonas sp. RRj228 (KACC91052) 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.