KR101091526B1 - Lysobacter antibioticus strain having antifungal activity isolated from soil and uses thereof - Google Patents

Abstract

The present invention relates to a method for assaying the antimicrobial activity of the microorganism Lysobacter antibioticus HS124 strain isolated from the rhizosphere and using it as a microbial agent, and to the environmental pollution, agricultural products, and It is an environmentally friendly alternative to address the side effects of pesticide accumulation. More specifically, the conditions for inhibiting the growth of phytopathogens secreted by HS124 were purified, and the culture conditions were set to maximize the antibacterial ability of the HS124 strain for use as a microbial agent. In addition, the culture conditions at the onset was set on the basis of nutrients that can be easily obtained for self-cultivation in general farms, and the microbial antimicrobial activity at this time was excellent in inhibiting pepper blight.

Phytopathogenic fungi, antifungal activity, lysobacter antibiotics, microbial agents

Description

Lysobacter antibioticus strain having antifungal activity isolated from soil and uses according to the antifungal activity isolated from soil

The present invention relates to a lysobacter antibioticus strain having antifungal activity isolated from soil and its use, and more particularly, to a lysobacter antibioticus strain having antifungal activity against phytopathogenic fungi, said strain Microbial preparation for phytopathogenic fungi control as an active ingredient, microbial preparation for phytopathogenic fungi control containing 4-hydroxyphenylacetic acid isolated from the strain, plant control by spraying an effective amount of the strain on the plant or soil Method, the phytopathogenic fungal control comprising culturing the strain in a medium containing an iron (III) compound, chitin powder, complex fertilizer (N, P ₂ O ₅ , K ₂ O), sucrose, yeast extract method for producing a microorganism preparation for, and iron (III) compound, chitin powder, compound fertilizer _{(N, P 2 O 5,} K 2 O), Rye can containing sucrose and yeast extract small Anti emitter Bio Tea Syracuse strain relates to a culture medium for.

Agriculture using existing chemical pesticides has been aimed at increasing the number of crops, but has many side effects such as environmental pollution, pesticide accumulation on soil, agricultural products, and killing, and the appearance of resistant pathogens. Therefore, amid growing concern about the adverse effects of pesticides, environmental awareness and environmental awareness are increasing, and environmentally friendly farming methods are being sought. This development of agricultural cultivation is the opportunity to secure domestic and foreign competitiveness, bring about the increase of agricultural income in Korea and further promote the eco-friendly agriculture aiming for high quality agricultural products.

At present, researches using microorganisms as well as by-products such as rice bran, food and crab shell are being actively conducted for eco-friendly agricultural production. Among them, farming methods using antagonistic microorganisms, which have excellent ability to control diseases in crops and promote crop growth and soil improvement effect, are widely spread. The main soil microorganisms used are Bacillus sp., Pseudomonas sp., And Rhizobium sp.

Lysobacter sp. Among these microorganisms are antagonistic microorganisms commonly found in the soil, and they grow well in the roots or leaves of plants and secrete many metabolites, causing Rhizoctonia solani , Fusarium oxysporum, Botrysis cinerea, Phytophthora capsici , and It is known to prevent nematodes and pests and to promote crop growth. Lysobacter metabolites that are primarily involved in inhibiting phytopathogens include quinoline compounds (Evans et al., 1978, The Journal of Antibiotics 31, 952-958), cetacandines A and B (Meyers et al., 1985, The Journal of Antibiotics 38, 1642-1648), cephabacin (Ono et al., 1984, The Journal of Antibiotics 37, 1528-1535), chitinase, β-1,3 & β-1,4- Glucanase, protease (Moataza, 2006, Research Journal of Agriculture and Biological Sciences 2, 274-281). Because of the antimicrobial activity of lysobacter that secretes these various active substances has been actively studied for a long time. However, research on the biologics of Lysobacter has only a rudimentary content on antimicrobial agents, and its use as a formulation is extremely rare at home and abroad.

As described above, the secondary metabolite secreted by Lysobacter genus acts as an important factor in inhibiting the growth of pathogens causing plant diseases, and when inoculated into crops, it can grow well in soil and crop roots. Considering the fact that it is expected, excellent efficacy as a biological product is expected.

The present inventors have already succeeded in the development of functional compost containing a large amount of chitin-decomposing microorganisms through the isolation, identification and in-flight experiments to identify the characteristics of chitin-decomposing microorganisms. The plant disease control effect and the plant growth promoting effect of the functional compost containing was confirmed. In addition, efforts have been made to disseminate farms through technology transfer.

The present invention has been made in accordance with the requirements as described above, the present inventors have studied to identify new antimicrobial actives from the HS124 strain, as a result of confirming the antimicrobial activity in the microbial culture and extracted with ethyl acetate several times column chromatography The structure was analyzed by purification and secretion of the enzyme was confirmed. In addition, the secretion of low molecular weight side pore (siderphore) binding to iron (III) was confirmed in HS124 culture medium, the side pore secreted in the Lysobacter genus is a rare result. The present invention was completed by establishing culture conditions for maximizing the antimicrobial activity of microorganisms having such characteristics and using them as microbial agents.

In order to solve the above problems, the present invention provides a lysobacter antibioticus strain having antifungal activity against phytopathogenic fungi.

The present invention also provides a microbial agent for controlling phytopathogenic fungi containing the strain as an active ingredient.

The present invention also provides a microbial agent for controlling phytopathogenic fungi containing 4-hydroxyphenylacetic acid isolated from the strain.

The present invention also provides a method for controlling plant diseases by spraying an effective amount of the strain on plants or soil.

In addition, the present invention is phytopathogenic comprising culturing the strain in a medium containing an iron (III) compound, chitin powder, complex fertilizer (N, P ₂ O ₅ , K ₂ O), sucrose, yeast extract Provided is a method for preparing a microbial agent for fungal control.

The present invention also provides a culture medium for lysobacter antibioticus strain comprising an iron (III) compound, chitin powder, complex fertilizer (N, P ₂ O ₅ , K ₂ O), sucrose and yeast extract do.

According to the present invention, various secondary metabolites secreted by HS124 play an important role in inhibiting the growth of the pathogenic fungus P. capsici and are advantageous in nutrient contention through sidepore secretion that chelates iron (III). HS124 as a microbial agent that adds iron (III) in an amount equivalent to 100 μM to maximize the activity of HS124 can be usefully used for crop protection in farms.

In order to achieve the object of the present invention, the present invention provides a Lysobacter antibioticus strain having antifungal activity against phytopathogenic fungi. The strain is more preferably Lysobacter antibioticus HS124 (KACC 91438P). In the Lysobacter antibioticus strain of the present invention, the phytopathogenic fungi are Phytophthora capsici , Fusarium oxysporum , Diaporthe citri , Pycurularia Pyricularia grisea , or Rhizoctonia solani , but is not limited thereto.

The Lysobacter antibioticus strain of the present invention may further comprise at least one enzymatic activity selected from the group consisting of chitinase, β-1,3-glucanase, lipase, protease and gelatinase. And, more preferably, it may include all of chitinase, β-1,3-glucanase, lipase, protease and gelatinase activity.

The Lysobacter antibioticus strain of the present invention is also characterized by secreting a catechol type sidephore.

The present invention also provides a microbial agent for controlling phytopathogenic fungi comprising the lysobacter antibioticus strain of the present invention as an active ingredient. The microbial agent is characterized in that the iron (III) compound is added at a concentration of 100μM, the plant disease may be pepper blight, but is not limited thereto.

The present invention also provides a microbial agent for controlling phytopathogenic fungi containing 4-hydroxyphenylacetic acid isolated from the lysobacter antibioticus strain of the present invention. The microbial agent is characterized in that the iron (III) compound is added at a concentration of 100μM, the plant disease may be pepper blight, but is not limited thereto.

The present invention also provides a method of controlling plant diseases by spraying an effective amount of the lysobacter antibioticus strain of the present invention on a plant or soil. In the method of the present invention, the plant disease may be pepper blight, but is not limited thereto.

In another aspect, the present invention, the lysobacter antibioticus strain of the present invention is a medium containing an iron (III) compound, chitin powder, complex fertilizer (N, P ₂ O ₅ , K ₂ O), sucrose, yeast extract It provides a method for producing a microbial agent for phytopathogenic fungal control comprising the step of culturing in. In the method of the present invention, the concentration of the iron (III) compound is preferably 100 μM and the plant disease is preferably pepper blight.

The present invention also provides a culture medium for lysobacter antibioticus strain comprising an iron (III) compound, chitin powder, complex fertilizer (N, P ₂ O ₅ , K ₂ O), sucrose and yeast extract do. The concentration of the iron (III) compound in the medium is preferably 100 μM.

Hereinafter, the present invention will be described in detail.

The present invention relates to a microbial preparation of myosphere microorganisms having antimicrobial activity. More specifically, the present invention relates to the characteristics of the antimicrobial activity strong myosphere microorganisms isolated from farms in Naju, Korea, and its application as a microbial agent using the same. Secondary metabolites secreted by antagonistic microorganisms directly or indirectly inhibit the growth of fungi and microorganisms, nematodes, which cause plant diseases. Therefore, the present invention relates to a microbial agent having an excellent effect on identifying the metabolites related to disease inhibition using the microorganisms, and promoting crop growth as well as the ability to suppress crops when processed in a crop as a microbial agent.

In the present invention, microorganisms having strong chitin and antimicrobial activity were isolated from the microorganisms separated from the soil, and the microbial culture was extracted with ethyl acetate and purified several times through column column chromatography. Among the purified fractions, fractions having antibacterial activity against Phytophthora capsici were provided by structural analysis through HPLC and CG-MS.

In addition, HS124 secreting enzymes (chitinase, β-1,3-glucanase, lipase, protease) among the substances that inhibit the growth of pathogens are provided through microbial culture and selection medium.

In addition, the microbial culture was extracted with ethyl acetate and purified several times through column column chromatography and HPLC for separation of the side pore, an iron chelate compound that is advantageous in iron (III) nutrient contention with pathogens. And it is provided via ¹ H NMR to identify the OH group chelating iron (III) in the skeleton of the side pore.

In addition, in order to maximize the antimicrobial activity of the strain, chitin powder, complex fertilizer (N, P ₂ O ₅ , K ₂ O), sucrose, yeast extract was added to the culture medium containing various concentrations of iron (III), By measuring the concentration of microorganisms, antimicrobial activity, side pore activity and the like to investigate the iron (III) concentration of the optimum conditions to provide the optimum culture conditions.

In addition, in order to determine the effect of controlling pepper blight through pepper pot experiment, culture medium (C1) cultured HS124 containing 100 μM of iron (III), culture solution (C2) cultured HS124 without iron (III), And using the respective medium (M1 and M2) that did not culture microorganisms as a control to provide a microbial agent for the control of pepper blight.

The present invention analyzes microbial metabolites involved in disease control by newly separating microorganisms having strong chitin decomposition ability in soil, and studies the establishment of culture conditions as microbial agents, crop disease control effects, and growth promoting effects.

The microorganism is known as Plant Growth Promoting Rhizobacteria (hereinafter referred to as "PGPR"), which has a function of inducing plant resistance and promoting growth, and is Gram-negative, bacilli, and lysobacter sp. The strain Lysobacter antibioticus is named HS124.

The present invention isolates, purifies, and identifies substances that inhibit the growth of pathogenic fungi in HS124 strain culture. Specifically, the supernatant obtained by centrifugation of the HS124 strain culture solution was extracted with ethyl acetate (Ethyl acetate) and the concentrated concentrate was subjected to Diaion HP-20, silica gel, Sephadex LH-20 column chromatography, etc. Purify. In addition, HPLC, GC-MS instrumental analysis for the material structure analysis.

The antimicrobial active substance 4-hydroxyphenylacetic acid isolated from the HS124 strain through this process destroys the mycelia of the pathogenic fungus P. capsici at a concentration of 100 ppm.

In addition, chitinase and β-1,3-glucanase were identified in the microbial culture supernatant. The secretion of lipase, protease and gelatinase was shown in skim milk medium and 1% Tween. It is confirmed in LB medium and gelatin medium to which 80 is added.

Purification of the side pore, another substance involved in the antimicrobial activity of the HS124 strain of the present invention was also through the process of purifying the antimicrobial active substance, and at each step, side pore active fractions were obtained through side pore liquid analysis and Arnow assay. Through the structure was confirmed. The strain secreted a catechol type iron chelating compound, sidepoa at a concentration of about 10 ppm and provided excellent iron (III) binding capacity.

The first embodiment of the method of using the HS124 strain of the present invention as a microbial agent is to find the optimum culture conditions in the minimal nutrient medium. To incubate, incubate with chitin powder, yeast extract, and FeCl ₃ · 6H ₂ O, respectively, at 0, 50, 100, 150, and 200 μM concentrations. At this time, the reason for providing iron can be understood that the microorganisms used in the present invention have a high iron (III) binding force, and benefit from their growth by using iron (III) as a nutrient. Therefore, the addition of various concentrations of iron (III) provides optimum iron utilization conditions. Other studies have also reported that iron (III) plays an important role in biological control by promoting the secretion of secondary metabolites, which are involved in pathogenesis (Keel et al., 1989, Phytopathology 79, 584-589; Van Rij et. al., 2004, The American Phytopathological Society 17, 557-566).

The second embodiment of the method of using the HS124 strain of the present invention as a microorganism and a formulation shows that the antimicrobial activity of the microorganism is excellent when the iron (III) equivalent to 100 μM is added. Incubate conditions by adding potassium and sucrose.

Specifically, chitin powder, yeast extract, nitrogen (N), phosphoric acid (P ₂ O ₅ ), potassium (K ₂ O) and sucrose (sucrose) and FeCl ₃ · 6H ₂ O 100 μM were added based on 1 L of culture. By culturing. In this case, nutrients, except for chitin powder and yeast, were added on the 3rd day of culture, and the main purpose is to prevent the contamination of other microorganisms and to grow only by selecting culture of HS124 strain with excellent chitin decomposition ability when grown in farms. . In addition, the added nutrients provide sufficient nutrients to plants as well as the growth of microorganisms.

A third embodiment of the method of using the HS124 strain of the present invention as a microbial agent is to test the antimicrobial activity of the microbial culture inoculum before the pot experiment. As a result, even when only 30% of the culture inoculum is added under unsterilized conditions, growth of pathogenic fungi is completely inhibited. More preferably, the present microbial agent may be used when diluted in water when used in a real farm, so that the control effect of pathogens can be maintained.

In the fourth embodiment of the method of using the HS124 strain of the present invention as a microbial agent, the effect of disease control is examined through a pot test. The concentration of red pepper disease pathogens in pepper plant experiments was 1 × 10 ⁶ zoospore / ml, and 30 ml of microbial cultures were used per 600 g of pot. The treatment was used to culture the microorganisms as a control medium (C2) without the addition of iron (III) and the control medium (C2) without the addition of iron (III) to the nutrient, which is a method using HS124 as a microbial preparation. It is used as a medium (M1) without addition of iron (III) and a medium (M2) without addition of iron (III).

As a result of experiments using the HS124 strain of the present invention as a microbial agent, the control group (M1 and M2) was wilted with all pepper diseased 5 days before the inoculation of the pathogen, the incidence of the disease is higher than 80%. However, the incidence of disease in the microbial culture C1 treatment was 20%, which is significantly lower than that of the control, and there is a big difference in growth. The results showed that chitinase and β-1,3-glucanase activity measured in the rhizosphere as microbial activity indicators were higher than M1 and M2 in soils treated with C1 and C2. do.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Isolation of HS124 Strain from Root Soil

The microorganisms used in the present invention were obtained by collecting soil from Naju farms and using a dilution plate method (chitin medium 0.5%; Na ₂ HPO ₄ 0.2%; KH ₂ PO ₄ 0.1%; NaCl 0.5%; NH ₄ Cl 0.1%). MgSO ₄ · 7H ₂ O 0.05%; CaCl ₂ · 2H ₂ O 0.05%; yeast extract 0.05%; agar 2%; pH 7.0) to isolate a single strain forming a strong clear zone. CP medium for secondary separation (colloidal chitin 0.25%; Na ₂ HPO ₄ 0.1%; KH ₂ PO ₄ 0.05%; NaCl 0.25%; NH ₄ Cl 0.05%; MgSO ₄ 7H ₂ O 0.025%; CaCl ₂ 2H ₂ O 0.025%; yeast extract 0.025%; agar 2%; potato dextrose broth 0.5%) and cultured with pathogenic fungi, one of the most potent strains was isolated.

Then, mixed with 25% glycerol solution for use in strain antimicrobial activity test and metabolite test was stored at -70 ℃ and 16S rRNA sequence analysis for strain identification.

The isolated microorganism was identified as Lysobacter antibioticus by 16S rRNA sequencing and named HS124. The strain was deposited with the Korea Agricultural Microbial Resources Center (KACC) on February 2, 2009 (Accession No .: KACC 91438P).

1 is a colony-forming picture of HS124 smeared on chitin medium, and this strain strongly inhibited the growth of Pytophthora capsici , a causative agent of pepper disease (FIG. 2), in addition to inhibit the growth of various phytopathogenic fungi as shown in Table 1 Could.

Table 1.Regarding various phytopathogenic fungi of Root-microbe L. antibioticus HS124 isolated in Example 1 Inhibitory ability.

Antifungal activity is less than 10% inhibition (-), 10-20% inhibition (±), 20-50% inhibition (+), 50-80% inhibition (++), 80-100% inhibition (+++). Reported.

Example 2 Isolation and Identification of Antibacterial Substances from HS124 Strains

The supernatant (15 L) is prepared by centrifugation by culturing the microorganisms in Luria-Bertani (LB) medium for 4 days. The pH of the supernatant was adjusted to pH 3 with stock HCl, extracted with ethyl acetate and concentrated to obtain crude. Diaion HP-20 column chromatography was used to separate and purify the material, and antimicrobial materials were eluted from high to low polarity using water and methanol (100: 0 ~ 0: 100, v / v). Each fraction was tested for antimicrobial activity by replacing the pathogenic fungus P. capsici with paper disk method, and silica gel column chromatography was performed with 70% and 100% methanol fractions showing excellent activity. The mobile phase was ethylacetate: methanol (100: 0, 50:50, 0: 100, v / v) and purified once more with silica gel, showing the strongest antimicrobial activity in the 100% ethanol fraction and the mobile phase was hexane: ethylacetate: methanol (20:40:40, v / v / v) was used. Finally, the antibacterial material was separated by Sephadex LH-20 column chromatography, and the mobile phase was 100% methanol. In order to confirm that it is a single material, it was analyzed by high performance liquid chromatography (HPLC) with a C ₁₈ reversed phase column and the antimicrobial material was detected at 210 nm with a UV-VIS detector. The structural formula of the material was characterized by gas chromatography-mass spectrometry (GC-MS).

As a result, 4.5 g crude was obtained when the HS124 strain culture solution (15 L) was extracted with ethyl acetate. Antibacterial material isolated through purification was detected as a single substance by HPLC analysis, and inhibited the growth of P. casici even in a small amount of 2 mg as shown in FIG. The isolated pure antibacterial material was identified as 4-hydroxyphenylacetic acid by GC-MS analysis (FIG. 4).

Example 3 Antimicrobial Agents Are Pathogenic Fungi P. capsici Effect on Mycelial Growth of

The isolated antimicrobial substance was prepared by dissolving in methanol at 500 ppm concentration, and treated with 100 ppm and 200 ppm in P. capsici mycelia cultured in PDB medium for 7 days. The cells were incubated in a 27 ° C. incubator for one day and observed morphological changes of mycelia through an optical microscope.

As a result, there was no change in the form of mycelia without 4-hydroxyphenylacetic acid. However, when 100 ppm and 200 ppm 4-hydroxyphenylacetic acid were incubated with the mycelia, it was folded and decomposed as shown in FIG. , An abnormal form of destruction, etc.

<Example 4> Degradation enzyme secreted by the HS124 strain

To investigate the chitinase activity, 0.05 ml of the supernatant of HS124, 0.5 ml of 0.5% colloidal chitin, 0.45 ml of 50 mM sodium acetate buffer (pH 5.0) were mixed and reacted at 37 ° C. for 1 hour. After 1 hour, 200 μl of 1 N NaOH was added and reacted in boiling water for 15 minutes, followed by centrifugation to obtain a supernatant. 750 μl of the supernatant was mixed with 1 ml of Schales'reagent (0.5 M sodium carbonate and 1.5 mM potassium ferricyanide), and colorimetrically measured at 420 nm using N-acetyl glucosamine as the standard.

β-1,3-glucanase activity was added to 50 μl of HS124 supernatant, 50 μl of 10 mg / ml laminarin, and 400 μl of 50 mM sodium acetate buffer (pH 5.0) to react at 37 ° C. for 1 hour. After the reaction, 1.5 ml of 3,5-dinitrosalicylic acid (DNS) was added thereto, boiled for 5 minutes, and colorimetric measurement was performed at 550 nm using glucose as a standard.

Protease and gelatinase secretion was determined by milk agar medium (1 L water; yeast extract 1.5 g; agar 15 g; skim milk powder (Campina, Belgium) 100 g), gelatin medium (1 L water; gelatin 10 g; MgSO ₄ 7H ₂ O 0.4 g; NaCl 1 g; CaCl ₂ · 2H ₂ 0 0.2 g; KCl 0.2 g; NH ₄ NO ₃ 1.5 g; KH ₂ PO ₄ 0.5 g; K ₂ HPO ₄ 0.5 g; Yeast extract 1.5 g; Agar It was confirmed that the clear zone is formed by incubating the HS124 in 15 g), the lipase secretion was confirmed by culturing HS124 in LB solid medium to which 1% Tween 80 is added to form the cells.

As a result, the HS124 strain showed high chitinase activity on day 4 of culture and then gradually decreased. The maximum production chitinase amount was 3.98 units / ml. β-1,3-glucanase activity produced up to 1.79 units / ml on day 3 and then decreased. In addition, protease and gelatinase secretion was also confirmed by forming a clear zone in each selection medium, and lipase was also confirmed through the growth of the HS124 strain in the selection medium.

Table 2. Types of enzymes and enzyme activities secreted by L. antibioticus HS124 strain.

+: Shows enzyme activity

a: Chitinase and β-1,3-glucanase activity were investigated from the supernatant of L. antibioticus .

b: Production of lipase, protease and gelatinase was detected on each selection medium.

<Example 5> Side pore secretion confirmation from HS124 strain

100 μl of HS124 culture was inoculated into CAS medium, followed by incubation for one day, and the secretion of the side pore was confirmed by changing the color of the medium from blue to orange. In addition, the Arnow assay (Arnow, 1937, Journal of Biological Chemistry 118, 531-537) and the Csaky assay (Csaky, 1948, Acta Chemica ) were used to determine whether the secreted sidepoa was of the catechol or hydroxamate type. Scandinavica 2, 450-454).

To purify the side pores from the HS124 culture, the microorganisms were extracted with ethyl acetate, and the crude was concentrated by diaion HP-20 and silica gel column chromatography. Each fraction was analyzed by Arnow and Sidepoa liquid analysis (Schwyn and Neilands, 1987, Analytical Biochemistry 160, 47-56) to confirm the presence of the side pore. Only active peaks out of the three peaks collected were collected in fractions. The collected side pore active material was subjected to ¹ H NMR (300 MHz FT-NMR Spectrometer) to identify the OH group binding iron (III).

HS124 strain secreted a catechol type sidepore having a high iron binding capacity of about 10 ppm in the culture. After extraction and purification of the culture, three peaks were detected at 270 nm by UV-VIS detector by HPLC analysis, and side pore reaction was observed at 9.15 minutes by side pore liquid analysis. Only the peak was fractionated and a large amount of OH group bound to iron (III) was confirmed at around 12 ppm by ¹ H NMR analysis.

Example 6 Optimal Growth Conditions by Addition of Various Concentrations of Iron ( III) at Minimum Medium Condition of HS124 Strains

HS124 strain was added with various concentrations of iron (FeCl ₃ · 6H ₂ O; 0, 50, 100, 150, 200 μM) in minimal medium (1 L of water; 1.5 g of crab shell powder; 0.03 g of yeast extract; pH 7.0) Incubate for 5 days and count the bacteria in LB solid medium by dilution plate method. In addition, 100 μl of each culture solution was inoculated into CAS medium to determine the size of side pore activity. Each culture supernatant is added to PDA medium by 30% sterilization or non-sterilization for 15 minutes at 120 ℃. P. capsici mycelia pre-cultured in PDA for 7 days were cut to 6 mm in diameter and placed in the center immediately after the mixed medium (30% + PDA of supernatant) was solidified and incubated for 5 days, and the diameter of the mycelia was measured. Each control group used a mixed medium made by adding only 30% of medium without culturing bacteria, and the antimicrobial activity was calculated by the following formula.

Antimicrobial Activity (%) = (A-B) / A x 100

A: Mycelial length of control

B: Mycelial Length of Medium Added with HS124 Supernatant

As a result, when the HS124 strain was cultured in a minimal medium containing 100 μM of iron, the number of bacteria was the highest as 8.9ｘ10 ⁸ , and when more iron (III) was added, the number of bacteria decreased. Side pore activity was also proportional to the orange halozone size up to 100 μM iron and decreased at higher concentrations (Table 3). In addition, as a result of the antimicrobial activity assay of each culture, the culture medium of the HS124 strain added 100 μM of all the sterile or non-sterile conditions showed the highest value of about 20%, 33% (Fig. 8). Therefore, the concentration of iron (III) suitable for culturing HS124 strain in the future was set to 100 μM to proceed with future experiments.

Table 3. L. antibioticus HS124 microbial count and side pore activity with various concentrations of iron (III).

a: counted in LB agar medium by dilution plate method.

b: Measured by the size of halozone, reported as less than 1 mm (±), 3-4 mm (++), less than 7 mm (+++).

Example 7 Preparation of Microbial Preparation of Cultured HS124 Strains by Addition of Nitrogen, Phosphoric Acid, and Potassium

Cultivate the HS124 strain in medium of the following composition (1 L; crab shell powder 1.5 g; yeast extract 0.03 g; sucrose 2 g; N 0.42 g; P ₂ O ₅ 0.34 g; K ₂ O 0.34 g; pH 7.0). At this time, iron (FeCl ₃ · 6H ₂ O, 27 mg) added (C1) or not added (C2), and the control medium without the inoculation of microorganisms without iron (M1) or medium without addition ( M2). Each culture supernatant incubated for 5 days under the above conditions is added to the PDA medium 15%, 30%, 50% by sterilization or non-sterile for 15 minutes. Immediately after the medium was hardened, P. capsici mycelia pre-cultured in PDA for 7 days were cut out 6 mm, incubated in mixed medium (microbial supernatant + PDA) for 5 days, and the diameter of the mycelia was measured.

As a result, it is shown in FIG. 9 that C1 has stronger antibacterial activity than culture medium C2 to which iron (III) was not added under all conditions in which the culture medium was sterilized or non-sterile. In particular, it was possible to completely inhibit the growth of the mold as a result of mixing and testing the PDA culture to 30% without sterilizing the C1 culture.

Example 8 Inhibitory Effect of HS124 Microbial Agents on Pepper Disease

Pepper seeds ( Capsicum annuum L.) are planted in a 3ｘ3 cm plastic tray with topsoil, and 4 weeks later, red pepper seedlings are planted in 600 g pots. At 2, 3, 4, and 5 weeks after the red pepper preparation, 30 ml of M1 and M2 were treated with the microorganism preparation C1 prepared above and C2 as a comparative example, and as a control. At 6 weeks, 30 ml of P. capsici resident (1ｘ10 ⁶ zoospore / ml) was inoculated into every pot and sampled at 0, 2, 4, 6, and 8 days of inoculation of pathogens to determine the mortality and morbidity of the live body and root. Investigate.

The mortality of roots was added to 500 mg of the sampled roots, 10 ml of 0.6% 2,3,5-triphenyltetrazolium chloride in 0.05 M phosphate buffer (pH 7.4) and washed in distilled water after reaction at 30 ° C. for 24 hours and at 70 ° C. Extracted in 95% ethanol for 4 hours and the absorbance is measured at 420 nm. And the disease incidence rate is represented by calculating the percentage of the total number of peppers that the disease occurred in each treatment.

C1 as a microbial preparation prepared in Example 7, C2 used as a comparative example, and each control group M1 and M2 were treated to pepper as in the procedure of FIG. Within 5 days of pathogen inoculation, the peppers of the control (M1 and M2) rapidly became ill and began to wither and their live weight also decreased rapidly (FIGS. 11 and 12). However, the microbial treatments (C1 and C2) continued to increase their live weight up to 8 days after sampling despite the pathogen inoculation. As a result of investigating chitinase and β-1,3-glucanase activity of microorganisms in rhizosphere, microbial treatments C1 and C2 showed higher values than control (FIG. 13). In addition, in all the treatments, considering the root mortality rate and disease incidence was found to have a control effect of about 70% or more in the pepper of the C1 treatment (Fig. 14 and Fig. 15). The test was repeated three times in all.

1 is a photograph of colony formation in chitin medium of L. antibioticus HS124 strain isolated from the present invention.

2 shows P. capsici inhibitory ability of L. antibioticus HS124.

Figure 3 shows the P. capsici inhibitory ability of the antimicrobial material isolated from L. antibioticus HS124 strain culture.

4 shows GC-CI (a) and GC-EI (b) spectra of 4-hydroxyphenylacetic acid, an antimicrobial agent isolated from L. antibioticus HS124 strain culture.

5 shows the morphological changes of P. capsici hyphae with treatment of 4-hydroxyphenylacetic acid (a: control, b: 100 ppm treatment, c: 200 ppm treatment).

Figure 6 shows the HPLC spectra of the side pore active fractions isolated from L. antibioticus HS124 strain culture.

Figure 7 shows the ¹ H NMR spectrum of the side pore isolated from L. antibioticus HS124 strain culture.

Figure 8 shows the antimicrobial activity against P. capsici according to sterile, non-sterile of L. antibioticus HS124 strain culture medium with various concentrations of iron (III).

Figure 9 shows the antimicrobial activity against P. capsici in sterile or non-sterile conditions of 100 μM iron (III) addition (C1) and without (C2) L. antibioticus HS124 strain culture.

10 shows an experimental design for verifying the effect of the control of pepper blight of L. antibioticus HS124 strain.

Figure 11 shows the test results of pepper blight control effect according to C1, C2, M1 and M2 treatment.

Figure 12 shows the ground and underground live weight results of pepper according to C1, C2, M1 and M2 treatment.

Figure 13 shows chitinase and β-1,3-glucanase activity in musculosphere soil following C1, C2, M1 and M2 treatment.

Figure 14 shows the results of pepper root mortality according to C1, C2, M1 and M2 treatment.

15 shows the results of pepper blight incidence according to C1, C2, M1 and M2 treatment.

Claims (18)

Lysobacter antibioticus strain having antifungal activity against phytopathogenic fungi. The Lysobacter antibioticus strain according to claim 1, which is Lysobacter antibioticus HS124 (KACC 91438P). The method of claim 1, wherein the phytopathogenic fungi are Phytophthora capsici , Fusarium oxysporum , Diaporthe citri , Pyricularia grisea and Lysobacter antibioticus strain, characterized in that the selected from the group consisting of Rhizoctonia solani . The lysobacter according to claim 1, further comprising at least one enzymatic activity selected from the group consisting of chitinase, β-1,3-glucanase, lipase, protease and gelatinase. Antibioticus strains. The lysobacter antibioticus strain according to claim 1, which secretes a catechol type sidephore. A microbial agent for controlling phytopathogenic fungi, comprising the Lysobacter antibioticus strain of claim 1 as an active ingredient. The microbial agent according to claim 6, wherein the iron (III) compound is added at a concentration of 100 µM. 7. The microorganism preparation according to claim 6, wherein the plant disease is pepper disease. A microbial agent for controlling phytopathogenic fungi, comprising 4-hydroxyphenylacetic acid isolated from the lysobacter antibioticus strain of claim 1 as an active ingredient. 10. The microbial agent according to claim 9, wherein the iron (III) compound is added at a concentration of 100 µM. 10. The microorganism preparation according to claim 9, wherein the plant disease is pepper disease. A method of controlling plant diseases by spraying an effective amount of the lysobacter antibioticus strain of any one of claims 1 to 5 on a plant or soil. The method of claim 12, wherein the plant disease is pepper blight. The lysobacter antibioticus strain according to any one of claims 1 to 5 is used as an iron (III) compound, chitin powder, complex fertilizer (N, P ₂ O ₅ , K ₂ O), sucrose and yeast extract. Method for producing a microbial agent for phytopathogenic fungi comprising the step of culturing in a medium containing. The method of claim 14, wherein the concentration of the iron (III) compound is 100 μM. 15. The method of claim 14, wherein the plant disease is pepper disease. The lysobacter antibioti according to any one of claims 1 to 5, comprising an iron (III) compound, chitin powder, complex fertilizers (N, P ₂ O ₅ , K ₂ O), sucrose and yeast extract. Culture medium for cous strain. 18. The medium according to claim 17, wherein the concentration of the iron (III) compound in the medium is 100 µM.

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