KR100391802B1 - Method of biological control for Grass Large Patch Disease and Cucumber Damping off Disease by using Helicosporium sp.(KCTC0635BP) - Google Patents

Abstract

The present invention relates to a plant control method using an antifungal Helicosporium strain, and the LCLK99 (KCTC 0635BP) strain of Helicosporium sp. It has an excellent antimicrobial effect on the pathogen of large patch disease.

Description

Method of biological control for Grass Large Patch Disease and Cucumber Damping off Disease by using Helicosporium sp. (KCTC0635BP)}

The present invention relates to a biological control method using a novel antifungal Helicosporium sp. More specifically, the present invention relates to a method for biological control against grasshopper patch disease and cucumber mortar disease using antifungal Helicosporium sp. Strain (KCTC0635BP).

Recently, antagonistic microorganisms are separated from natural ecosystems such as soil, and microorganisms are directly or planted to control plant diseases, and antifungal substances secreted by them are used for control (Omura et al. 1988, Mandeel and Baker 1993, etc.). Putton and Peng 1993) have been studied for biological control methods. The biological bangjebeop is Bacillus (Bacillus) genus Pseudomonas (Pseudomonas) in such a bacteria and tricot Delmas (Trichoderma), A penny room Solarium actinomycetes, such as fungi and Streptomyces (Streptomyces) in such (Penicillium) in use of In Korea, researches on plant diseases control using these microorganisms have been made in various ways.

Therefore, the present invention is based on the results of the above assay for the antimicrobial activity of phytopathogenic bacteria of the LCLK99 strain (KCTC0635BP) of the genus Helicosporium sp. ( Hlicosporium sp.) Having antimicrobial activity against Rhizoctonia Solani It is to provide an effective plant control method by the novel antagonistic strains.

The object of the present invention was achieved by identifying new antagonistic fungi purely from chestnut trees, identifying them, and analyzing the antimicrobial and plant growth effects of Rhizoctonia Solani .

Hereinafter, the configuration and operation of the present invention will be described in detail by way of examples.

1 is a photograph showing the color change of the strain of the present invention Helicosporium sp. Helicosporium sp. LCLK99 (KCTC0635BP) before and after incubation in PDA medium.

Figure 2 is a photograph showing the vial cells of the present invention strain Helicosporium sp. Helicosporium sp. LCLK99 (KCTC0635BP) shaken cultured in PD broth medium.

Figure 3 is an optical micrograph of the strain of the present invention strain Helicosporium ( Helicosporium sp.) LCLK99 (KCTC0635BP).

Figure 4 is an electron micrograph of the strain of the present invention Helicosporium sp. Helicosporium sp. LCLK99 (KCTC0635BP).

5 is a photograph showing the spores of LCLK99 (KCTC0635BP) genus of the present invention strain Helicosporium sp.

Figure 6 is a photograph showing the antagonistic effect of LCLK99 (KCTC0635BP) of the present invention strain Helicosporium sp.

The present invention comprises the steps of separating and identifying the LCLK99 strain (KCTC0635BP) of the present invention strain Helicosporium sp. LCLK99 strain (KCTC0635BP) of the present invention strain Helicosporium sp. ( Hlicosporium sp.) Was selected as Rhizoctonia solani (cucumber), Rhizoctonia solani AG2-2, Fusarium oxysporium ( Fusarium oxysporium ), Phytopthora dreschsler ( Phytopthora dreschsler ) while replacing the culture in the flora culture disc to measure the low support (Clean zone) to investigate the antimicrobial effect; The present invention isolates helicase course Four Solarium (Helicosporium sp.) Belongs to a PDA medium containing a culture filtrate of LCLK99 (KCTC0635BP) with various concentrations of Rhizoctonia small Raney (Rhizoctonia solani) (cucumber), Rhizoctonia small Raney (Rhizoctonia solani ) After inoculation with AG2-2 and Fusarium oxysporium , the mycelial growth was measured to investigate the antibacterial effect of the culture filtrate concentration of LCLK99 (KCTC0635BP) strain of the present invention Helicosporium sp. Doing; The mycelial growth after immersing the Rhizoctonia solani bacterium in a PDA medium containing various concentrations of the culture filtrate of the strain of Helicosporium sp. Helicosporium sp. Investigating the antimicrobial effect by concentration and immersion time of the culture filtrate of the present invention Helicosporium sp. (LCL9999 (KCTC0635BP) strain culture filtrate Helicosporium sp.); The present invention consists of spraying the culture filtrate of the strain LCLK99 (KCTC0635BP) in the genus Helicosporium sp. Helicosporium sp.

Hereinafter, specific methods of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Isolation and Identification of Microorganism Strains of the Invention

Chestnut branches that seemed to have died from chestnut pulmonary disease in Yangbo-myeon, Yangbo-myeon, Hadong-gun, Gyeongnam, South Korea were collected and surface sterilized with 75% alcohol in the room, and then separated using water agar (WA). The edges of the cultured bacteria in water agar medium were removed and inoculated in potato medium (Potato dextrose agar: PDA) and incubated in a 25 ° C thermostat. As a result, as shown in FIG. 1, there was no pigment production before culturing in potato medium, but after culturing, a dark brown pigment was produced. In FIG. 2, inoculation in PD broth medium was incubated with shaking (120 rpm / min). ) Shows the cells of the isolated strain of the present invention showed a grape grain shape. As a result of observing the pure strain isolated from the cultured strain with an optical microscope of 400 times (Olympus, Japan), it showed a cell shape as shown in Figure 3 and the form observed with an electron microscope of 380 times is shown in Figure 4. In addition, spores of the isolated strain of the present invention showed a bacterium form (400-fold) as shown in FIG. The present invention isolates the synthesis the results were classified as helicase course Four Solarium sp (Helicosporium sp.) According to the guide of the Father (Bergy's manual) was named Heli course Four Solarium (Helicosporium sp.) In LCLK99. The pure isolated strain of the present invention was deposited with the Accession No. KCTC 0635BP to the Biotechnology Research Institute which is an international microorganism deposit institution.

On the other hand, the proliferation amount according to the temperature of the strain of the present invention is shown in Table 1 and the growth temperature was around 30 ℃.

Cell growth rate by temperature Temperature (℃) Mycelial diameter growth (mm) 4 days 6 days 8th 12 days 20 12.3 18.3 21.7 35.3 25 22.7 34.0 43.3 61.7 30 30.0 41.0 51.7 73.3

Example 2 antibacterial effect of the strain of the present invention

The present invention Heli Course Four Leeum (Helicosporium sp.) In LCLK99 (KCTC0635BP) to investigate whether the antagonistic effect against plant pathogenic strains Heli Course Four Leeum (Helicosporium sp.) In LCLK99 (KCTC0635BP) strains 5mm colony disc (disc ) Was inoculated in PDA medium and incubated at 25 ° C. for 5 days, followed by Rhizoctonia solani (cucumber), Rhizoctonia solani AG2-2, Fusarium oxysporium , Phytopthora dreschsler mycelial disk was incubated at a distance of 40mm to measure the clean zone.

As a result, as shown in FIG. 6 of the present invention, the LCLK99 strain (KCTC0635BP) of the genus Helicosporium sp. (KCTC0635BP) is Rhizoctonia solani isolated from cucumber and lycyanonia sorani isolated from grass. ( Rhizoctonia solani ) showed very high antagonistic activity against AG2-2. Low support for R. solani (cucumber) was 15 mm and R. solani AG2-2 was 12 mm, respectively. This is summarized in Table 2.

Antimicrobial Effects of LCLK99 (KCTC0635BP) Strains of Helicosporium sp. Fungus host Antimicrobial activity Coiling Lysis Over growing Low support Rhizoctonia solani cucumber - + - 15 Rhizoctonia solani AG2-2 grass - + - 12 Fusarium oxysporium cucumber - + - 5 Phytopthora dreschsler Torreya - - - -

Example 3: Inhibitory effect on phytopathogens by concentration of culture filtrate

In order to measure the inhibitory effect on phytopathogens by concentration of the culture filtrate of the strain of the present invention, 500 ml of PD broth was put into a plastic container for 850 ml bottle mushroom cultivation and sterilized for 30 minutes at 1.5 atm. Inoculated with one total flora disk (10mm) incubated in PDA medium for 7 days and incubated for 10 days in a 25 ℃ shake incubator (120rpm) and sterilized for 30 minutes at 121 ℃, 1.5 atm. The mycelia were removed with Kimwips to obtain a culture filtrate (crude filtrate). The culture filtrate was added to the PDA medium at 1, 5, 10, 20, 50, 100%, and sterilized, and then 20 ml of the 9 cm Petri dishes were dispensed. Inoculated with Rhizoctonia solani (cucumber), Rhizoctonia solani AG2-2, Fusarium oxysporium mycelial disk (5 mm) incubated in PDA medium for 5 days Mycelial growth was measured. Each treatment was repeated three times.

Culture of results, heli Course Four Leeum (Helicosporium sp.) In LCLK99 (KCTC0635BP) and is found to be highly confrontation culture upon antagonism of plant pathogens, heli Course Four Solarium in (Helicosporium sp.) LCLK99 (KCTC0635BP ) The inhibitory effect of the filtrate added by concentration was shown in Table 3. The concentration of LCLK99 (KCTC0635BP) in Helicosporium sp. Showed a significant difference in the inhibition rate according to the phytopathogens. Among them, the inhibition rate for turbid patch disease was the highest, showing 100% inhibition rate even at 5% concentration. Rhizoctonia solani showed more than 70% inhibition at 5% concentration and 100% inhibition at 50% concentration.

In the case of Fusarium oxysporium , the inhibitory effect was lower than that of Rhizoctonia solani with 66.7% inhibition rate at 100% concentration.

The present invention isolates helicase course Four Solarium (Helicosporium sp.) The culture filtrate in the LCLK99 (KCTC0635BP) Rhizoctonia small Raney (Rhizoctonia solani) (cucumber), Rhizoctonia small Raney (R. solani), and Fusarium AG2-2 Effect of Fusarium oxysporium on Growth Inhibition % Culture (added to PDA) R. solani (cucumber) R. solani AG2-2 Fusarium oxysporium Diameter (mm) Control (%) Diameter (mm) Control (%) Diameter (mm) Control (%) 0 63.3 - 28.3 - 15.0 - One 48.7 23.1 26.3 7.1 16.0 0.0 5 17.3 72.7 0.0 100 16.0 0.0 10 11.3 82.2 0.0 100 14.0 6.7 20 6.3 90.1 0.0 100 14.0 6.7 50 0.0 100 0.0 100 11.0 26.7 100 0.0 100 0.0 100 5.0 66.7

Example 4: Lysatonia sorani by concentration and immersion time of the culture filtrate ( Rhizoctonia solani Antimicrobial effect on

The culture filtrate of the culture filtrate of the present invention Helicosporium sp. Helicosporium sp. (KCTC0635BP) of the culture filtrate to determine the antimicrobial effect according to the concentration, soaking time for Rhizoctonia solani bacteria 1 , 5, 10, 20, 50, 100% by adjusting the ratio of 20ml to 50ml Erlenmeyer flasks were sterilized. Rhizoctonia solani cultured in PDA medium (5mm) was immersed for 1, 4, 8, 24 hours, then the culture filtrate was removed with sterile water and inoculated in PDA medium and incubated in a 25 ℃ thermostat. Mycelial growth was measured.

As a result, the antimicrobial effect according to the culture filtrate concentration and soaking time of LCLK99 (KCTC0635BP) strain of Helicosporium sp. The longer the soaking time, the higher the suppression rate at low concentrations, 1 hour at 100% concentration, 3 hours at 50% concentration, and 100% inhibition at 24 hours immersion at 20% concentration.

The present invention isolates helicase course Four Solarium (Helicosporium sp.) In concentrations, antibacterial effect of immersion time LCLK99LCLK99 (KCTC0635BP) density(%) Immersion time (hr) Diameter (mm) Rachonia Sorani (R. solani) Control (%) 2 days 4 days 2 days 4 days 0 14824 27.027.127.827.5 85.085.085.085.0 ---- ---- One 14824 27.026.024.724.0 656261.752.0 0.04.111.212.7 23.527.127.438.8 5 14824 26.026.027.025.7 60.062.060.059.1 3.74.12.96.6 29.427.129.430.5 10 14824 25.223.024.424.7 53.058.059.258.0 6.715.122.210.2 37.731.830.431.8 20 14824 16.00.00.00.0 43.00.00.00.0 40.7100100100 49.4100100100 50 14824 0.00.00.00.0 0.00.00.00.0 100100100100 100100100100 100 14824 0.00.00.00.0 0.00.00.00.0 100100100100 100100100100 Control (%) = (1-treatment / check) X 100

Example 5: Control effect of grasshopper patch disease using strain culture filtrate of the present invention

In order to investigate the control effect of turfgrass patch disease using LCLK99 (KCTC0635BP) culture filtrate in the present invention strain Helicosporium sp. Helicosporium sp. From May to June 1999, Dongnae Benest golf course No. 11 Joysia Grass The filtrate was inoculated in 1 ×, 10 ×, 150 ×, and 500 × cultured in PD broth. After the treatment, the size of the lesion was measured, and the edges of the lesion were marked in four directions. After treatment, the lesion size was measured. For the control effect, the progress of desease was determined by investigating the occurrence area before and after treatment.

As a result, 100% of the control filtrate of LCLK99 (KCTC0635BP) in Helicosporium sp. (KCTC0635BP) and 100% control showed 100% control value, which showed the same effect as montserene used as a control drug (Table 5). . In addition, in the sustained period after treatment, there was no translation until 30 days when the culture filtrate was treated, but the disease progressed after 30 days when the pesticide was treated.

Effect of Rhizoctonia solani AG2-2 on the Control of Turf Patch Disease of the Culture Filtrate of the Present Invention process Area of Patch (%) Control (%) Control 23.5 - 1 X 0.0 100 10 X 0.0 100 150 X 18.6 20.9 500 X 25.2 0.0 Control (%) = (1-treatment / check) X 100

Example 6 Effect of Strain Culture Filtrate of the Present Invention on Plant Growth

Experimental Example 1: Filter Paper Treatment

After adjusting the concentration of the strain culture filtrate of the present invention to 1, 5, 10, 20, 50, 100%, respectively, sterilized the seed (cucumber cucumber) seed for 6 hours at 121 ℃ 30 minutes and then washed with distilled water Ten pellets were placed in a disposable petri dish covered with one 9 cm filter paper, and 1 ml of distilled water was added thereto. Wrapped in a wrap and stored in a 25 ° C thermostat to prevent moisture evaporation. After 3 days, germination rate and root growth were examined and repeated 3 times with 10 grains per repetition.

Experimental Example 2: Pot Treatment

Cucumber seeds soaked in a filtrate of 15 cm in length and 6 cm in diameter in a plastic pipe sterilized in each concentration described in Experimental Example 1 were sown. The pipe was erected in a cylindrical transparent acrylic container with a 30 cm diameter lid, and then filled with 15% of water up to 10 cm in height to maintain a constant moisture in the pipe. After 7 days of sowing, germination rate and the length of the above ground and below ground were investigated.

Experimental results of the culture filtrate of LCLK99 (KCTC0635BP) in Helicosporium sp. Helicosporium sp. The germination rate of cucumber by filter paper treatment method was 100% in all concentrations. The growth of roots showed better growth at 1-20% concentration compared to no treatment, which gave nutrition to plant growth. At concentrations above 50%, however, growth was reduced compared to no treatment. There was no significant difference in the growth of ground rods when planting in pots, but the underground part (root) was better than untreated at 5% or more.

Cucumber growth by concentration of culture filtrate by filter paper treatment density(%) Root Length (cm) Germination rate (%) No treatment (distilled water) 19.6 100 One 29.3 100 5 34.3 100 10 38.3 100 20 33.1 100 50 10.4 100 100 3.0 100

Cucumber growth according to the concentration of the culture filtrate in the pot density(%) Growth amount (mm) Germination rate (%) Underground Ground No treatment (distilled water) 12.6 3.8 100 One 11.3 4.7 100 5 16.0 3.6 100 10 14.5 3.2 100 20 16.6 4.7 100 50 17.4 3.4 100 100 16.0 2.0 100

As described above through the examples and experimental examples of the detailed description of the present invention, the present invention has the effect of providing a novel strain of the genus Helicosporium sp. Helicosporium sp. LCLK99 (KCTC0635BP) with excellent antibacterial activity against phytopathogens . have. In addition, the present invention is a very useful invention in the pesticide industry because it has an excellent effect of providing a biological pesticide effective for plant growth.

Claims (2)

In the biological control method for controlling grasshopper patch disease using antagonist microorganism or its culture filtrate, Method for biological control of turbid patchworm, characterized by using the strain LCLK99 (KCTC 0635BP) of the genus Helicosporium sp. In the biological control method for controlling omozoalok disease using antagonist microorganism or its culture filtrate, Method for biological control of cucumber omozalococcal disease, characterized by using the strain LCLK99 (KCTC 0635BP) of the genus Helicosporium sp.