KR100347721B1 - Antagonistic soil bacterium Pseudomonas fluorescens and biological control using the same - Google Patents

Abstract

The present invention relates to Pseudomonas fluorescens 2112 ( Pseudomonas fluorescens 2112) and a biological control method using the same. By inhibiting RNA synthesis and protein synthesis of phytopathogenic fungi by producing sex antibiotics and siderophores, they inhibit the growth of phytopathogens and maintain phytopathogenic activity, especially at acidic pH and high temperature. Also has excellent effects to maintain its activity.

Description

Antagonistic soil bacterium Pseudomonas fluorescens and biological control using the same

The present invention relates to Pseudomonas fluorescens 2112 (Accession No. KCTC 8944P) and a biological control method using the antagonist strain Pseudomonas fluorescens 2112. More specifically, the present invention relates to a novel antagonist Pseudomonas fluorescens 2112 for producing antibiotics and sideropores to effectively control phytopathogens and a biological control method using the same.

Everyone has acknowledged that organic synthetic chemical pesticides, which have been rapidly developed since the early 1950s, have contributed greatly to the world's food production, but serious damages such as the destruction of ecosystems due to its abuse are continuing. It is strengthening day by day. Under these regulatory conditions, the growth trend of the chemical pesticide market is gradually decreasing, while the market for new biopesticides and microbial products is becoming active.

Agricultural antibiotics using active substances secreted by microorganisms are mostly used by actinomycetes among soil microorganisms, and were developed relatively later than medicinal antibiotics. Among them, it was most developed especially in Japan. In 1958, Blastin for control of rice fever was known as the first commercial product.

In Korea, biocontrol methods using some imported microorganisms have been attempted, but most of them have not been able to perform their activities due to lack of adaptability to the climate climate and soil environment. Therefore, there is a need for a biocontrol method that selects and utilizes indigenous antagonistic microorganisms of Korean soil that can easily adapt to the environment of our region and become a predominantly restoration in the soil.

The loss of crop production by plant pests accounts for 12% of the total crop production worldwide, but only 20% of them are damaged by soil nematodes and bacteria, and most of them are caused by phytopathogenic fungi. Therefore, the necessity of controlling fungal phytopathogens is much more urgent than the necessity of controlling plant diseases by bacteria. Until now, organic chemical pesticides for fungal control have been mainly used, but there are limitations to continue use.

The microbial control method has the advantages of no environmental pollution, low toxicity, low production cost, and low investment in production equipment, and the research and development cost is about 1/10-1 / 20th of the existing pesticide development cost, and the development period is about 1/3 There is an advantage in carrying.

Siderophores, known as antagonists of antagonism, are water-soluble, low-molecular, ferric iron-chelating compounds, typically yellow-green fluorescent chromophores linked by peptide chains. chromophore). It is divided into two structural forms according to the composition and size of the peptide chain. It is a derivative of catechol (ο-dihydroxybenzene), which is a catechol amide (phenolates, catecholates) form and hydroxamic acid (hydroxamic acid). A derivative of acid (RCONHOH) is in the form of hydro-xamates. The catechol type siderophores include enterrobactin, parabactin, acrobactin, anguibactin, vibriobactin, and azotochelin. Siderophores in the form of hydroxamate include ferrichrome, ferrioxamine, coprogen, and nocardamine. Recently, citrate-hydroxamate forms of siderophores include arthrobactin, aerobactin, schizokinen, and quinoline. Pseudo-bactin and pyoverdin are known as siderophores of the form.

The amount of iron (Fe) required by microorganisms in the natural environment is about 0.4 to 4.0 μM for Gram-positive bacteria and fungi, and 0.3 to 1.8 μM for Gram-negative bacteria. Most of this iron is in a neutral or alkaline pH under aerobic environment dissolution constant of 10 ^-38.7 so insoluble ferric-exist as oxyhydroxide polymer [ferric-oxyhydroxide polymer [Fe ( OH 3)], the free ferric iron The concentration of free ferric iron is generally very low, with a dissolution constant of ^10-18 at pH 7.

Although iron is abundant in the soil with 1 to 6% of the total inorganic ions, the amount of iron ions (Fe ³⁺ ) required by microorganisms is not sufficient to maintain their growth and will be a competitive factor among all soil microorganisms. There is no choice but to. Thus, in order to overcome this low-iron stress, many aerobic and breathable anaerobic microorganisms are required by a special iron regulated outer membrane protein (IROMP) to effectively obtain sufficient iron for growth. It has a high affinity iron transport system, which releases a low-molecular soluble siderophore that is highly compatible with iron ions and has a fluorescent color (400-2,000 daltons). After the formation of the protein, it binds to a recognate-specific receptor on the outer membrane and is sequentially introduced into the cell through the cytoplasmic membrane by an energy-dependent process. Sidepore biosynthesis and outer membrane proteins associated with these high affinity iron transport systems are controlled by iron ions.

Iron is generally an essential factor for spore germination and germ tube elongation of phytopathogens. If iron ions are not confined, the growth of plant pathogens may be active, resulting in plant root infiltration and infection. Cause. Thus, PGPR in the rhizosphere perches on the surface of plant roots and inhibits the growth of phytopathogens by establishing an advantage in the competition of iron ions absorption, which are mostly sideropores that secrete fluorescent pseudomonads. Siderophore inhibits spore germination and germ tube elongation of phytopathogens by chelating iron around the roots. Therefore, this competitive antagonism reduces soil infectious diseases and promotes rooting ability of host plants, which is a very effective biological control method to improve crop growth and increase yield.

Therefore, the present inventors have isolated and selected strains having a strong antagonistic activity against Phytophthora capsici , a major causative agent of red pepper disease, and investigated the physical properties of antifungal antibiotics, which are antagonists produced by this strain, We tried to investigate the control mechanism.

An object of the present invention is to provide an antagonistic strain Pseudomonas fluorescens 2112 which produces antifungal antibiotics and sideropores to strongly inhibit phytopathogens. Another object of the present invention to provide a biological control method using the antagonistic strains.

The object of the present invention is to identify antibiotics and Pseudomonas fluorescens after separating the antagonistic strain Pseudomonas genus 2112, which inhibits the development of pepper blight bacteria by producing antibiotics and side pores from the soil of low-pest farmland, which performs natural agriculture. The antimicrobial activity of the antibiotics produced by this strain was investigated by the paper disc method, and then the effects of pH and temperature on the antibiotic antimicrobial activity were investigated. Subsequently, in the production of antibiotics, the present invention antagonist Pseudomonas fluorescens 2112 investigated the effects of carbon source, nitrogen source, and inorganic salt to find the optimum production conditions, and then extracted and purified antibiotics produced. Using the strain and labeling the radioisotope with a precursor, the antimicrobial mechanism of the extracted antibiotic was investigated, and the antimicrobial activity was confirmed by in vivo biocontrol experiments using red pepper as a host plant. And this was achieved by purifying the siderophore produced by the antagonistic strain Pseudomonas fluorescens 2112 of the present invention, and analyzing and identifying the amino acids.

Hereinafter, the configuration and operation of the present invention.

1 is a degree of growth inhibition of the phytophthora capsici ( Phytophthora capsici ), which is the causative bacterium by Pseudomonas fluorescens 2112 of the present invention according to the pairing culture method on PDA ( Pseudomonas fluorescens 2112) Figure is a picture.

Figure 2 is a photograph showing an orange halo formed by the sideropore produced by the present invention antagonist Pseudomonas fluorescens 2112 in CAS medium.

Figure 3 is a photograph of observing the form of the antagonistic strain Pseudomonas fluorescens 2112 of the present invention with a fluoroscopic electron microscope.

Figure 4 shows the fatty acid analysis results for the identification of the present invention antagonist Pseudomonas fluorescens 2112.

5 is Biolog of the present invention antagonistic strain Pseudomonas fluorescens 2112 ^? It shows the result of identification by identification system.

FIG. 6 is a photograph showing the inhibition zone of the phytotoktor capsicum strain by the antibiotic of the present invention antagonist Pseudomonas fluorescens 2112 in a PDA plate medium.

Figure 7 is a photograph showing the antifungal activity of antibiotics produced by the antagonist Pseudomonas fluorescens 2112 of the present invention against the growth of Phytophthora capsicum mycelium.

8 is a photograph showing the inhibition of phytophthora cap seed by culturing the antagonist Pseudomonas fluorescens 2112 of the present invention in broth on PDB.

9 shows the HPLC pattern of antibiotics produced from the present invention antagonist Pseudomonas fluorescens 2112.

Figure 10 is a photograph of the antibiotic activity produced from the antagonist Pseudomonas fluorescens 2112 of the present invention using a phytophthora capsicum strainer.

Figure 11 shows the Fab-mass spectrum of the antifungal antibiotics obtained from the present invention antagonist Pseudomonas fluorescens 2112.

12 shows the ¹ H-NMR spectrum of the antifungal antibiotics produced from the present invention antagonist Pseudomonas fluorescens 2112.

Figure 13 shows the ¹³ C-NMR spectrum of the antifungal antibiotics produced from the antagonist Pseudomonas fluorescens 2112 of the present invention.

14 is a photograph showing the results of in vivo confirming the antifungal activity of Pseudomonas fluorescens 2112 of the present invention in the growth of red pepper.

15 is a flowchart illustrating the purification process of the table Aberdeen ₂₁₁₂ (pyoverdin ₂₁₁₂₎ produced from the present invention gilhanggyun main Pseudomonas fluorescein sense 2112.

16 is a Sephadex G-25 column (Sephadex G-25 column) shows the dissolution results of the present invention gilhanggyun main Pseudomonas fluorescein sense 2112 produces a table ₂₁₁₂ Aberdeen purified by.

FIG. 17 shows UV absorbance spectra of Fyberdine ₂₁₁₂ and their ferric complexes purified from the present invention antagonist Pseudomonas fluorescens 2112 in pH 6.5 aqueous solution.

The present invention isolates the Pseudomonas strains from indigenous antagonistic bacteria isolated from low-disement cropland soil, which performs natural farming, and then produces antibiotics among these Pseudomonas strains to prevent the development of pepper blight, while producing sideropores. Final selection of the antagonistic strains; Pseudomonas fluorescens 2112 of the selected antagonist strain Pseudomonas fluorescens 2112 was examined by gram staining and microscopy to examine its morphological characteristics, and also by using biochemical properties and API tests, fatty acid analysis, and Biolog's identification system. Identifying the strain of the present invention with Pseudomonas fluorescens; Culturing the antagonist Pseudomonas fluorescens 2112 of the present invention in King's B broth to obtain antibiotics, and then examining the antimicrobial activity of the antibiotics with a free disk method; Investigating the pH stability of the antibiotic produced by the antagonist strain of the present invention by measuring the antagonistic activity against the bacteriostatic bacterium after adjusting the antibiotic solution obtained from the antagonist Pseudomonas fluorescens 2112 to pH1 ~ 13; The antibiotics produced by the antagonist Pseudomonas fluorescens 2112 of the present invention were measured by measuring the antifungal activity against phytopathogens after treating the antibiotic control agent obtained from the present invention antagonist Pseudomonas fluorescein 2112 at a temperature range of 20 to 121 ° C. Examining the thermal stability of the; Adding 10 carbon sources in a Davis minial medium, culturing the antagonistic strains of the present invention, and measuring the growth of the growth bacteria to investigate the effect of the carbon source on the production of antibiotics; Investigating the effect of nitrogen source on the production of antibiotics by adding the carbon source and 13 kinds of inorganic and organic nitrogen sources showing the optimal antibiotic production conditions in the Davis minimum medium and culturing the antagonistic strain of the present invention and measuring the growth of the growth bacteria; Investigation of the effect of salt on antibiotic production by adding the carbon source, nitrogen source and 12 salts showing the optimal production of antibiotics to Davis Davis medium and culturing the antagonist Pseudomonas fluorescein 2112 of the present invention Making; Extracting antibiotics by adding n -butanol to the culture medium of the antagonist Pseudomonas fluorescens 2112 and shaking the mixture to concentrate the antimicrobial active material under reduced pressure, and then fractionate with ethyl acetate to extract antibiotics; Dissolving the ethyl acetate extract in methanol and then performing Diaion HP-20 column chromatography; Sepadex LH-20 column chromatography of the active fraction obtained by Diaion HP-20 column chromatography again; Extracting the antifungal active substance by developing the active fraction obtained by Sephadex LH-20 column chromatography on silica gel TLC; HPLC purification of the sample obtained by silica gel TLC to verify the purity; Phytophthora capsicum, a bacteriostatic bacterium, was tested according to the method of Hideyo Yamagachi using [ ³ H] leucine, [ ³ H] adenine, [ ¹⁴ C] glucose, and the radioactivity was measured. Identifying the antagonistic mechanism of antibiotics produced by the antagonist Pseudomonas fluorescens 2112; Investigating the onset inhibitory power of antibiotics produced by the antagonist Pseudomonas fluorescens 2112 of the present invention to the phytophthora cap C. Culturing the antagonistic strain Pseudomonas fluorescens 2112 of the present invention in SM medium to produce a siderophore; The ferric siderophore of the produced siderophore was first purified and then de-ironed, followed by purification of pure siderophore using CM- Sephadex C-25 and Sephadex G-25, followed by TLC. Confirming; Analyze the amino acid of the purified siderophore to identify the structure.

Example 1: Isolation of Antagonist Strains

In this example, 10 ^-2 in sterile saline solution is used as a source of separation of low-pest agricultural soils of natural agriculture using indigenous antagonists of Gyeongju Ahwa area to select antagonistic microorganisms that have strong dominant, indigenous and reversible ability in local soil. Suspended and diluted until, and inoculated with 0.1 ml each of the smear rod in LB agar medium, and incubated for 1 day at 30 ℃. In order to isolate and cultivate the antibiotic-producing Pseudomonas genus, Pseudomonas isolation agar (PIA) was isolated using King's B agar. ( F. oxysporum) and P. capsici ( P. capsici) are strains that form a large inhibitory distance by replacing them in PDA to isolate and select antagonistic strains that can be controlled by antibiotic production. Was separated. In order to isolate Siderophore-producing strains, most siderophore-producing strains produce fluorescens, producing antibiotics from sucrose minimal agar (SMA). Strains that generate fluorescens were selected for the strains to isolate large and clear orange rings on CAS (chrome azurol S) blue agar plate medium. Amicon centriprep was applied to the bacteria isolated from antibiotic-producing indigenous antagonists for the final selection of low-molecular-product indigenous antagonists such as antibiotics. The antagonism of low molecular weight collected by 10 (MW 10,000) was determined by phytopathogenic activities such as F. solani, F. oxysporum and P. capsici. The strains were selected by investigating them by pairing culture test, cell mass test and the like. In addition, since many of the antibiotics were heat-resistant, the antagonistic force after heat-treating the culture supernatant of the isolated antagonist bacteria at 80 ° C. for 30 minutes was also examined by a cell mass test, and most of the antibiotics were butanol. n considering that the oil-soluble to be eluted in a non-polar solvent-extracted with butanol (n -butanol), were examined the way the drag force test cell mass (cell mass test). In addition, for the selection of siderophore-producing indigenous antagonist strains among antibiotic-producing antagonists, CAS was isolated using SM medium and CAS medium. The strains with the best siderophore production capacity were finally selected using the rate and siderophore activity assay by Arnow phenolic assay and Csaky hydroxamate assay. As a result, 120 species of Pseudomonase genus were isolated from PIA medium, which is a medium that isolates Pseudomonas genus from indigenous antagonistic bacteria isolated from low-cultivated soil of natural farming. Phytophthora capsici ( Phytophthora capsici ) and the cultivation and growth quantitative measurement method was performed to isolate the antagonistic bacteria antagonistic to the causative bacterium. The production of fluorescens in SMA media was also confirmed in the antagonistic bacteria that form a strong development zone against P. capsici . These isolates were isolated from strains that strongly produced siderophore in CAS for siderophore detection, which produced a large orange discoloration ring and was the largest antagonist to the phytophthora capsici . Indigenous antagonistic microorganisms PLP 2112 with final selection (Fig. 1, 2, Table 1).

Selection of indigenous antagonistic strains for phytophthora cap seed Strain Pairing distance (mm) Side low pore production (mm) ^* Cell volume inhibition (%) BLP-3034 6 0 72.7 BLP-5057 7 0 84.2 BLP-8117 6 0 87.5 BLP-4045 5 0 64.8 BLP-5042 8 5 56.5 BLP-6036 3 13 85.4 PLP-4059 5 9 75.4 PLP-2071 6 11 85.4 PLP-5046 One 7 68.1 PLP-2112 9 8 89.8 * Diameter of the ring formed by the shell sidelopor on the CAS agar plate

Example 2 Identification of Inventive Antagonist Strains

API for Taxonomic Identification of Pseudomonas Genus 2112 Strain Isolated from Example 1 Test (bioMerieux), Biolog's identification system (MicroLog ^TM 3), and various biochemical properties were used, and the morphology was observed by gram staining and transmission electron microscope (TEM). Based on this, the final strain was identified using the Bergey's Manual of Determinative Bacteriology index. As a result, the strain Pseudomonas genus 2112 of the present invention was identified as Gram-negative bacillus, and as a result of observing with an electron microscope to confirm its micromorphism, it was found to be bacilli having two to three flagella (FIG. 3). In addition, various biochemical test and API Pseudomonas fluorescens biotype F ( Pseudomonas fluorescens biotype F) was found to be 58.3% compared to the Pseudomonas ( Pseudomonas) identification method, such as test (Table 2) and fatty acid analysis (Fig. 4). It was shown to be ductile and confirmed using Biolog's identification system. As a result, Pseudomonas fluorescens biotype F ( P. fluorescens biotype F) showed 74.5% inductance (Fig. 5) and finally Pseudomonas fluore Sense biotype F or its related species was identified.

The identified Pseudomonas fluorescens 2112 strain of the present invention was deposited with KCTC 8944P on June 8, 1999 at the Biotechnology Research Institute.

Identification of antagonistic strain Pseudomonas fluorescens 2112 against phytophthora capsicum exam PLP Pseudomonas Fluorescence 24 hours 48 hours APItest Potassium Nitrate + + ± Tryptophan + + - Glucose - - - Arginine - - + Urea - - - Escalin + + + gelatin + + ± p-nitro-phenyl-β D-galactopyranoside - - - Glucose + + + Arabinos + + + Mannose + + + Mannitol + + + N-acetylglucosamine + + ± Maltose - - - Gluconate + + + Caprate + + + Adipate - - - Malate + + + Citrate + + + Phenyl-acetate - - - Tetramethyl-p-phenylenediamine + + + Cell formation rod rod Gram strain - - Single parameter One One Spore production - - Fluorescent dyes + + Pseudomonas fluorescein [Note] ±: 30 ~ 60% response

Example 3: Antimicrobial Activity of Antibiotic Produced by the Antagonist of the Present Invention

Inoculation of King's B broth in order to obtain an antifungal antibiotic from the antagonist Pseudomonas fluorescens 2112 of the present invention was incubated at 180 rpm for 4 days at 28 ℃. The centrifuged supernatant was recovered by centrifugation at 8,000 × g for 20 minutes, and the centrifugal supernatant was used for antifungal activity test. Antimicrobial activity was investigated by paper disc method. In other words, the spores of the phytophthora cap seed on the PDA medium were deposited on a 6 mm diameter disc and sterilized with an antimicrobial activity fraction sterilized for 10 minutes in a 100 ° C. water bath, followed by incubation at 28 ° C. for 2 to 4 days. We investigated the presence of antifungal inhibitory rings around the disc that inhibited mycelial growth. As a result of the experiment, a zoospore suspension of Capsicum phytophthora cap seeds was smeared onto PDA medium, and the disc paper on which the adjusted antibiotics were deposited was placed as a jersey germination germ of Pseudomonas cap seeds. Growth inhibition was shown around the disc paper (FIG. 6), and the Phytophthora cap seed on V8 juice medium _{2112 prepared} by mixing and dispensing the V8 juice medium with the adjusted antibiotics to confirm mycelial growth inhibition. Was inoculated in the center of the medium with a 6mm sized disk to check the growth of the hyphae. In addition, inoculated with Pitoptora cap seed grown in PDA medium with a 6mm sized disk in PDB and adjusted antibiotics were added at 30 ° C. Even when cultured, the mycelial growth of the disk inoculated with the disk could not be seen (FIGS. 7 and 8). Therefore, the antifungal antibiotics produced by Pseudomonas fluorescens 2112 were antifungal antibiotics that inhibited both the growth and the germination and the mycelial growth of Phytopytora cap.

Example 4 Characteristics of the Antibiotic Produced by the Antagonist of the Present Invention

Experimental Example 1: pH Stability

In order to examine the stability according to the pH of the antifungal antibiotics produced by the antagonistic Pseudomonas fluorescens 2112 of the present invention was carried out in a adjusted state. The stability to pH was adjusted to pH 1∼pH 13 with 1M HCl and NaOH, followed by pretreatment at 4 ° C. for 17 hours. After addition to the PDB, the antagonistic activity was examined by the growth cell mass measurement. As a result, as shown in Table 3, the antibiotic was found to be very stable under acidic pH but unstable in alkaline solution.

PH Stability of Antifungal Antibiotic Produced from Pseudomonas Fluorescens 2112 pH Relative Inhibition Rate (%) One 37.8 2 45.9 3 50.0 4 63.5 5 68.5 6 77.5 7 82.9 8 86.5 9 86.9 10 0 11 0 12 0 13 0 No antibiotic 0 None 100.0

Experimental Example 2: Thermal Stability

In order to examine the temperature-dependent stability of the antifungal antibiotics produced by the antagonist strain of the present invention was carried out in the adjusted state. Stability with respect to temperature was investigated by antimicrobial activity reduction method after 30 minutes (20 minutes in case of 121 degreeC) of the crude liquid at the temperature range from 20 degreeC to 121 degreeC. As a result of the experiment, as shown in Table 4, it was very stable to heat, and even after heat treatment for 30 minutes to 80 ℃, the antibacterial activity was maintained as it is. Therefore, it was confirmed that the antibiotic produced by the antagonistic strain of the present invention has a very stable property against heat.

Thermal Stability of Antifungal Antibiotics from Pseudomonas Fluorescens 2112 Temperature (℃) Relative Inhibition Rate (%) 40 86.5 60 83.3 80 50.5 100 0 121 0 No antibiotic 0 None 100.0

Example 5 Optimal Condition of Antibiotic Production of the Antagonist of the Invention

Experimental Example 1: Influence of Carbon Source

In the present experimental example, glucose was excluded from the Davis minimal medium, and 10 carbon sources were added at 1.0%, followed by culturing the present invention antagonist Pseudomonas fluorescens 2112 at 30 ° C. for 3 days. According to the antifungal activity was investigated. As shown in Table 5, glycerol showed the best effect on antifungal activity and growth of antagonistic bacteria. This is the same ingredient of King's B medium, which is the optimum medium in Pseudomonas. It was found to be an essential ingredient for the growth of the genus.

Effect of Carbon Sources on the Production of Antibiotics from Pseudomonas Fluorescens 2112 Carbon source Cell growth (Cell / mL) Inhibition Ratio (%) None 5.10 × 10 ⁷ 100.0 Arabinos 6.59 × 10 ⁸ 77.5 Saccharose 7.64 × 10 ⁸ 281.8 Glycerol 8.18 × 10 ⁸ 516.7 Glucose 6.39 × 10 ⁸ 155.0 Lamirarin 6.32 × 10 ⁷ 193.8 Galactose 6.63 × 10 ⁸ 67.4 Xylose 7.65 × 10 ⁸ 238.5 Lactose 6.09 × 10 ⁷ 124.0 Maltose 6.27 × 10 ⁷ 155.0 Fructose 6.91 × 10 ⁸ 182.4 [Note] The carbon source in the base medium [K ₂ HPO ₄ 0.7%, KH ₂ PO ₄ 0.2%, (NH ₄ ) ₂ SO ₄ 0.1%, sodium citrate 0.05% and MgSO ₄ · 7H ₂ O 0.01% pH 7.0] Pseudomonas fluorescens 2112 was incubated at 30 ° C. for 3 days.

Experimental Example 2: Influence of Nitrogen Source

In the present experimental example, the ammonium sulfate was removed from the Davis minimal medium, and the carbon source selected in the previous result was put in an optimal concentration, and then 13 kinds of inorganic and organic nitrogen sources were added in 0.5% increments, respectively. In the same way as in the case of antibiotics production was investigated. As shown in Table 6, it showed higher antifungal activity in the case of no addition of nitrogen source in beet extract, NaNO ₃ , yeast extract, tryptone, and pentose peptone. , Proteose peptone, a component of King's B medium, which is the optimum medium of Pseudomonas , showed high activity and growth rate, but showed less growth rate by tryptone, but more antifungal production It showed a lot of effects.

Effect of Nitrogen Sources on Antibiotic Production from Pseudomonas Fluorescens 2112 Nitrogen source Cell growth (Cell / mL) % Inhibition None 7.67 × 10 ⁷ 100.0 (NH ₄ ) ₂ SO ₄ 3.37 × 10 ⁸ 466.7 Urea 2.55 × 10 ⁸ 336.0 Bakto peptone 5.39 × 10 ⁸ 254.5 NH ₄ H ₂ PO ₄ 3.42 × 10 ⁸ 323.1 Malt extract 2.52 × 10 ⁸ 442.1 KNO ₃ 3.03 × 10 ⁸ 365.2 gravy 4.69 × 10 ⁸ 840.0 NaNO ₃ 2.97 × 10 ⁷ 700.0 Yeast extract 9.27 × 10 ⁸ 840.0 (NH ₄ ) ₂ S ₂ O ₈ 7.67 × 10 ⁵ 204.9 Trypton 7.85 × 10 ⁸ 1200.0 Proteose peptone NO. 3 8.18 × 10 ⁸ 289.7 (NH ₄ ) ₂ HPO ₄ 2.78 × 10 ⁸ 271.0 Pseudomonas after adding nitrogen source 0.1% to medium [K ₂ HPO ₄ 0.7%, KH ₂ PO ₄ 0.2%, sodium citrate 0.05%, MgSO ₄ .7H ₂ O 0.01% and glycerol 1.0%, pH 7.0] Fluorescein 2112 was incubated at 30 ° C. for 3 days.

Experimental Example 3: Effect of Inorganic Salts

In this experimental example, after adding the carbon source and nitrogen source determined in Experimental Example 1 and Example 2 at the optimum concentration to the Davis minimum medium except inorganic salts, 12 kinds of salts were added at a concentration of 5mM to produce antifungal antibiotics. The impact was investigated. As shown in Table 7, FeCl ₂ , LiCl and MgCl ₂ showed the best effect on antifungal activity and growth of antagonistic bacteria. In particular, LiCl grows about half of the antagonistic bacteria compared to other inorganic salts, but it shows an excellent antagonistic activity.

Effect of inorganic salts on the production of antibiotics from Pseudomonas fluorescens 2112 Mineral salts Cell growth (Cell / mL) % Inhibition None 3.46 × 10 ⁸ 100 FeCl ₂ 7.41 × 10 ⁸ 242.9 BaCl ₂ 2.34 × 10 ⁸ 130.8 RbCl 7.34 × 10 ⁸ 89.5 Na ₂ HPO ₄ 5.29 × 10 ⁸ 154.5 CaCO ₃ 6.35 × 10 ⁸ 51.5 ZnSO ₄ 2.01 × 10 ⁸ 56.7 MgCl ₂ 9.38 × 10 ⁸ 283.3 KCl 3.38 × 10 ⁸ 141.7 LiCl 3.65 × 10 ⁸ 340.0 K ₂ HPO ₄ 5.73 × 10 ⁸ 130.8 NaCl 9.97 × 10 ⁸ 94.4 CaCl ₂ 2.61 × 10 ⁸ 42.5 NOTE Mineral salts were added to the medium [yeast extract 0.5%, sodium citrate 0.05% and glycerol 1.0%, pH 7.0] and Pseudomonas fluorescens 2112 was incubated at 30 ° C. for 3 days.

Example 6: Isolation, Purification and Structural Analysis of Antibiotic Produced by the Antagonist of the Invention

First step: antibiotic extraction

In order to separate and purify the antimicrobial active material produced from Pseudomonas fluorescens 2112 of the present invention in large quantities, 9,000 mL of the culture solution was centrifuged at 4 ° C. and 8,000 × g for 20 minutes, and n had a large amount of antimicrobial activity in the supernatant. After adding the same amount of butanol and shaking, the n -butanol layer was separated and collected, which was a glass microfiber filter (Whatman , GF / A), and then the concentrated antimicrobial active material was concentrated under reduced pressure below 50 ℃. After recovering only the material dissolved in 100% methanol and concentrated under reduced pressure, and then dissolved it in the tertiary distilled water again insoluble material What. After filtering with 2 filter paper (ethyl acetate), an equal amount of ethyl acetate was added to recover the antibiotics dissolved in the ethyl acetate layer.

Second Process: Diaion HP-20 Column Chromatography

In this step, in order to isolate and purify the antimicrobial active material from the antibiotics obtained in the first step, the concentrated ethyl acetate extract was dissolved in methanol, and then loaded on a Diaion HP-20 column (2.5 × 80 cm), followed by methanol. (methanol) was allowed to flow 1 L at 0%, 50% and 100%. Each fraction was concentrated, dissolved in 10% methanol, and the antimicrobial activity was investigated using a paper disc method. The fractions showing antifungal activity were collected and concentrated under reduced pressure.

Third Process: Sephadex LH-20 Column

In this process, the active fraction obtained by Diaion HP-20 column chromatography was gel filtered by 10 mL per tube with methanol by LH-20 column (2.5 × 50 cm), and the antibacterial activity of each fraction was investigated. Fractions showing activity were collected and concentrated under reduced pressure, and the procedure was repeated twice. At this time, Sephadex LH-20 gel chromatography was carried out at a concentration of 10 minutes / mL under reduced pressure at 50 ℃, the antagonist was traced by testing the antagonism for each fraction.

Fourth Step: Extraction of Antifungal Active Substance by Silica Gel TLC

The active fraction obtained by Sephadex LH-20 column chromatography was developed by TLC with a developing solution in which n -butanol: acetic acid: water was 4: 2: 4, and the antifungal Rf value was about 0.89. Antifungal antibiotics were extracted from. The bands were collected and their antimicrobial activity was investigated to determine the degree of purification using HPLC.

Step 5: Confirmation of Purification of Antifungal Active Substance by HPLC

In this step, the purity of the sample obtained by silica gel TLC in the fourth step was verified by HPLC. The HPLC analysis conditions were LC-NH ₂ column, eluting at a rate of 11.0mL / min using 75% acetonitrile as elution solvent and the absorbance was measured at 260nm. As a result, the purification purity was verified as shown in FIG.

6th Step: Structure of Antifungal Active Substance

In this step, the activity of each of the antifungal active substances purified in the fifth step to confirm the degree of purification was carried out in PDA medium for phytophthora capsy's resident, and then antagonized in 6 mm disk after spores were spread on the medium. The material was absorbed and confirmed by the presence or absence of formation of a suppression ring around it (FIG. 10). Then analysis of the material structure is based on a report that Pseudomonas trick generated rayijo spear (Rhizosphere) rhizosphere plant biocontrol pathogenic fungi (biocontrol) showing the effect of the antifungal substance Eagle rusinol (acetylphloroglucinol) substance acetyl flow such In the mass spectrum (MS), it was found to have a molecular weight of 210, and showed an acetyl peak in ¹ H-NMR, and carbonyl carbon and acetyl in ¹³ C-NMR. Carbon peaks of -CH ₃ carbon, substituted aromatic ring of (CH 11) were observed (FIGS. 11, 12, 13). Pseudo having a molecular weight of 210 based on MS data and NMR data of roglucinol (acetylphloroglucinol) -based material and biomolecular NMR spectroscopy and Introduction to spectroscopy monas) -derived antifungal antibiotic 2, 4-diacetylphloro-glucinol (C ₁₀ O ₅ H ₁₀ ) was identified, the structure is shown in the following formula (I).

Example 7 Antagonistic Mechanism of Antibiotic Produced by the Antagonist of the Present Invention

In this example, the molecular mechanism of the antagonistic mechanism of phytopathogenic fungi using the purified antibiotics obtained from the antagonistic strain Pseudomonas fluorescens 2112 of the present invention was determined as the target strain of P. phytophthora capsicum. each ^[3 H] leucine (leucine), was tested by the ^[3 H] adenine (adenine), methods such as Hideyo Yamaguchi conducted using the ^[14 C] glucose (glucose) to proteins, nucleic acids, cell wall synthesis precursors to. In other words, 30 μl of purified antibiotics (controlled 50% methanol added) and 0.1 mL of the spore suspension of Phytoptotor Caps seed were inoculated in 0.85 mL of PDB diluted to 20%, and precultured at 30 ° C. for 1 hour, respectively. 20 μl of a labeled precursor of was added thereto, followed by shaking culture for 24 hours. The first investigation of [ ³ H] leucine was determined by the incorporation of the label leucine contained in a hot trichloroactic acid (TCA) -insoluble material. The reaction was carried out by adding the same volume of 10% TCA, heating at 90 ° C. for 15 minutes, and filtering the insoluble material contained in the reaction solution into a glass microfiber filter. , GF / A), washed twice, and air dried. The radio activity contained in this filter was measured. A second investigation of [ ³ H] adenine was determined by the incorporation of labeled adenin contained in cold TCA-insoluble (RNA plus DNA) material. After adding 1 mL of TCA, cold TCA-insoluble (RNA plus DNA) was collected with a glass microfiber filter, and another sample was added 1 mL 1N KOH. After 2 hours of treatment at 60 ° C, 1.8 mL of 20% cold TCA was added and precipitated, and then allowed to stand overnight at 0 ° C. The resultant was collected by a glass microfiber filter, and the radioactivity contained in the filter was obtained. (DNA) The third, [ ¹⁴ C] glucose irradiation was added 1 mL of 1N NaOH, decomposed in boiling water for 20 minutes and collected with a glass microfiber filter. It was washed twice with sterile water and air dry This was measured on a radioactivity contained in the filter was found to be a result, as can be seen in Table 8, the mechanism to inhibit the RNA synthesis inhibitors, and protein synthesis of pathogenic fungi.

Antifungal Mechanism of Pseudomonas Fluorescens 2112 in the Growth of Phytophthora Cap Cell composition Label Procursor Data (CPM) Control Treated with Pseudomonas fluorescens 2112 DNA and RNA [ ³ H] adenine 991,782 324,349 DNA [ ³ H] adenine 61,285 57,435 protein [ ³ H] leucine 2,644,208 335,252 Cell wall [ ¹⁴ C] glucose 35,805 37,439

Example 8: In vivo Port Control Experiment

In order to verify whether the antibiotic antimicrobial productivity antagonistic microorganisms exert control against phytopathogens in real soil, pepper experiments were carried out in a pot as a host plant, and soil soil: compost: sand 2: A mixture of 1: 1 was used. In the constant temperature and humidity room maintained at 28 ° C. and 70% humidity, three-leaved host peppers were transplanted into a pot of 15 cm in diameter and settled for 3 days, followed by cultivation of phytophthora cap seeds seeded in V8 juice agar medium. After recovering and inoculating 5 mL irrigation with 350 mL / mL incubator, the solution was treated for 1 day in a wet room (28 ° C., 70% humidity), and 5 mL of the selected control bacteria was treated with 6.0 × 10 ⁸ / mL of bacterial counts. Wet culture. The disease was periodically confirmed by raising it in a constant temperature and humidity room at 28 ° C. and 70%. At this time, it was confirmed that the control of the onset of pepper blight was compared to the control-free bacteria. As a result of the experiment, as shown in Figure 14, it was confirmed that the pepper bacterium bacterium Phytophthora capsi has excellent control.

Example 9 Purification and Characterization of Sidelopores Produced by the Antagonist Strains of the Invention

First Process: Side Lopore Production

In order to produce the sideropore from Pseudomonas fluorescens 2112 antagonist of the present invention was inoculated in SM medium and cultured for 3 days at 30 ℃ 180 rpm. The culture solution was centrifuged at 8,000 rpm for 20 minutes to recover the original needle supernatant, which was used.

Second Process: Sidelopor Purification

The siderophore produced in the first process was first extracted with a structurally stable ferric siderophore, and a single ferric siderophore with ion exchange chromatography using CM-Sephadex C-25. Purified and then deironed using this EDTA to purify pure siderophore using Sephadex G-25 and confirmed on TLC. As a result, the purified sidelopor was as shown in FIG. Confirmed that the iron is on, the addition of FeCl ₃ (100㎎ / ℓ) for 48 hours with shaking centrifuged supernatant of the culture broth by the use of SM medium for spore production medium in a restricted side represents the following dark red-brown, and CHCl ₃ / phenol Extraction was performed using (1: 1, v / w) and phenol was removed with ethyl ether. At this time, the crude Fe (III) siderophore complex (crude Fe (III) siderophore complex) was more effective than the crude siderophore (crude siderophore) is not extracted. It was found that this was because the solubility of the Fe (III) -siderophore complex in CHCl ₃ / phenol was increased. The extract was loaded on a CM Sephadex C-25 cation exchange column buffered with 0.05M sodium acetate buffer (pH 6.5) to show one high peak and two low peaks. Among them, the first peak was a reddish brown ferric siderophore complex, the second peak was a degradation product of the first peak, and the third peak was identified as a siderophor without iron binding. The first peak was subjected to secondary CM Sephadex C-25 cation exchange column chromatography to identify a single dark reddish brown peak, and the fraction obtained by eluting it was lyophilized and stored at -20 ° C. The lyophilized crude Fe (III) -siderophore complex was deironed at pH 4.0 with 5% 8-hydroxyquinoline. After de-ironing, the fluorescence was again confirmed, and two peaks of Sephadex G-25 gel filtration column chromatography were performed to obtain a single peak (FIG. 16).

Third Process: Identification of Sidelopores

The siderophore purified in the second step was hydrolyzed with 6N HCl and 47% HI, and analyzed by an amino acid analyzer. The maximum absorption wavelength of the purified siderophore was compared with that of the existing sideropore. Was identified. Since the siderophore produced by Pseudomonas fluorescein 2112 was found to be purified almost as a single substance through the purification process in the second process, the substance was identified through various analyzes such as amino acid analysis. It was named in Table Aberdeen ₂₁₁₂ (pyoverdin _2112). That is, the table verdin ₂₁₁₂ produced by Pseudomonas fluorescens 2112 exhibited a maximum UV absorption spectrum at 355-380 nm at pH 6.5, and the ferric-veverdin ₂₁₁₂ exhibited a maximum absorption spectrum at 390-405 nm ( 17). These results showed UV absorption wavelengths that closely matched the table verdin produced by most of the Pseudomonas spp. Known to date. Hydrolyzed purified table verdin ₂₁₁₂ with 6 N HCl to analyze the constituent amino acids, glycine (glycine), threonine (threonine), glutamic acid (glutamic acid), serine (serine), alanine (alanine), lysine (lysine) was identified and their composition ratio was 3: 2: 2: 1: 1: 1 (Table 9). The therefrom purification table Aberdeen ₂₁₁₂ is known UV maximum absorption spectrum similar to Pseudomonas lie to the side of producing Unlike pores of Perry baktin (ferribactin) or shoe gambling tin (pseudobactin) other and of Pseudomonas Table Aberdeen so far (spectrum) Considering the characteristics of Pseudomonas feverberdin, which is generally composed of about 6-12 peptides, the siderophore produced by the selected antagonist Pseudomonas fluorescens 2112 is known feverberdin It is considered to be in the form of a substance, but appears to be slightly different in terms of amino acid composition.

Constituents of Amino Acids Obtained from Hydrolysates of Fyoberdin 2112 Produced from Pseudomonas Fluorescens 2112 amino acid Mole% Composition ratio (%) Glycine 32.53 3 Threonine 25.03 2 Glutamic acid 18.16 2 Serine 12.28 One Alanine 9.54 One Lysine 12.89 One Note: A sample of Verberdin ₂₁₁₂ was obtained by hydrolysis in vacuo at 110 ° C. in 6N HCl for 24 hours.

As described above, the antagonistic strain Pseudomonas fluorescens 2112 of the present invention, isolated and identified from low-border cropland soil as described through the above examples and experimental examples, produces antifungal antibiotics and sideropores to synthesize RNA of phytopathogenic fungi. And by inhibiting the protein synthesis inhibits the growth of phytopathogens, especially in acidic pH and high temperature conditions, as well as maintaining the activity of inhibiting phytopathogens, in addition to the excellent effect of maintaining the activity in the soil, it is a very useful invention in the biopesticide industry.

Claims (2)

Antifungal antibiotics and sideropore productivity isolated from soil Antagonist Pseudomonas fluorescens 2112 (Accession No. KCTC8944P) A biocontrol method comprising the treatment of antagonistic strain Pseudomonas fluorescens 2112 according to claim 1 to red pepper to inhibit the growth of Phytophthora capsici .