Trichoderma asperellum T-5를 이용한 오이 모잘록병(Rhizoctonia solani)의 생물학적 제어 Biocontrol of Damping-Off(Rhizoctonia solani) in Cucumber by Trichoderma asperellum T-5원문보기
몇 년동안 게껍질이 풍부하게 있었던 밭토양에서 강한 키틴분해능력을 가진 Trichoderma 곰팡이를 분리하였다. 분리된 곰팡이의 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence 분석과 형태학적 특징을 살펴본 결과 Trichoderma asperellum으로 동정되었고, 이를 Trichoderma asperellum T-5 (TaT-5)로 명명하였다. 이 곰팡이는 chitianse와 ${\beta}$-1,3-glucanase같은 lytic enzyme을 분비하며, 키틴배지 상에서 6가지의 항 곰팡이성 물질을 생산했다. R. solani가 원인인 오이의 모잘록병에 대해 TaT 5의 방제효과를 보기 위해서 TaT-5 배양액(TA), chitin medium(CM), 증류수(DW)를 씨를 심은 후 10일 째에 각 pot에 관주했다. 그리고 관주 1주일 후에 R. solani의 균사를 갈아서 각 pot에 주었다. 실험기간 동안에 오이의 지상부와 지하부 생체중은 다른 처리구에 비하여 TA 처리구가 더 많이 증가하였다. 오이 잎에서 PR-protein (chitianse, ${\beta}$-1,3-glucanase) 활성은 R. solani 감염 후 CM과 DW에서 증가를 보였고, TA 처리구에서는 증가하다가 감소하는 경향을 보였다. 뿌리에서는 모든 처리구가 감소하는 경향을 보였지만 TA 처리구가 CM과 DW 처리구보다 감소하는 정도가 적었다. 오이의 잎과 뿌리에서 lignification related enzyme(POD, PPO, PAL)활성은 R. solani 감염 초기에는 증가하다가 점점 감소하는 경향을 보였다. 이러한 결과들은 TaT-5에 의하여 생산된 lytic enzymes와 항 곰팡이성 물질들이 오이에 R. solani의 공격을 줄여준다고 생각된다.
몇 년동안 게껍질이 풍부하게 있었던 밭토양에서 강한 키틴분해능력을 가진 Trichoderma 곰팡이를 분리하였다. 분리된 곰팡이의 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence 분석과 형태학적 특징을 살펴본 결과 Trichoderma asperellum으로 동정되었고, 이를 Trichoderma asperellum T-5 (TaT-5)로 명명하였다. 이 곰팡이는 chitianse와 ${\beta}$-1,3-glucanase같은 lytic enzyme을 분비하며, 키틴배지 상에서 6가지의 항 곰팡이성 물질을 생산했다. R. solani가 원인인 오이의 모잘록병에 대해 TaT 5의 방제효과를 보기 위해서 TaT-5 배양액(TA), chitin medium(CM), 증류수(DW)를 씨를 심은 후 10일 째에 각 pot에 관주했다. 그리고 관주 1주일 후에 R. solani의 균사를 갈아서 각 pot에 주었다. 실험기간 동안에 오이의 지상부와 지하부 생체중은 다른 처리구에 비하여 TA 처리구가 더 많이 증가하였다. 오이 잎에서 PR-protein (chitianse, ${\beta}$-1,3-glucanase) 활성은 R. solani 감염 후 CM과 DW에서 증가를 보였고, TA 처리구에서는 증가하다가 감소하는 경향을 보였다. 뿌리에서는 모든 처리구가 감소하는 경향을 보였지만 TA 처리구가 CM과 DW 처리구보다 감소하는 정도가 적었다. 오이의 잎과 뿌리에서 lignification related enzyme(POD, PPO, PAL)활성은 R. solani 감염 초기에는 증가하다가 점점 감소하는 경향을 보였다. 이러한 결과들은 TaT-5에 의하여 생산된 lytic enzymes와 항 곰팡이성 물질들이 오이에 R. solani의 공격을 줄여준다고 생각된다.
A fungal strain of Trichoderma having strong chitinolytic activity was isolated from field soil enriched with crabshell for several years. Based on 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence analysis and morphological characteristics, the fungus was identified as Trichoderma asperel...
A fungal strain of Trichoderma having strong chitinolytic activity was isolated from field soil enriched with crabshell for several years. Based on 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence analysis and morphological characteristics, the fungus was identified as Trichoderma asperellum and named as Trichoderma asperellum T-5 (TaT-5). The fungus released lytic enzymes such as chitinase and ${\beta}$-1, 3-glucanse, and produced six antifungal substances in chitin broth medium. To demonstrate the protective effect of TaT-5 against damping-off in cucumber plant caused by Rhizoctonia solani, TaT-5 culture broth (TA), chitin medium (CM) and distilled water (DW) were applied to each pot at 10 days after sowing, respectively. Then, the homogenized hyphae of R. solani were infected to each pot at 1 week after TaT-5 inoculation. During experimental period, fresh weight of shoot and root in cucumber plant more increased at TA treatment compared to other treatments. PR-proteins (${\beta}$-1, 3-glucanase and chitinase) activities in cucumber leaves markedly increased at CM and DW treatments, but the activity slightly increased and then decreased at TA treatment at 3 days after infection of R. solani. The activity of PR-proteins activities in cucumber roots at all treatments decreased with time where the degree of decrement was more alleviated at TA treatment than CM and DW. These results suggest that the lytic enzymes (chitinase and ${\beta}$-1, 3-glucanse) and antifungal substances produced by TaT-5 can reduce the pathogenic attack by R. solani in cucumber plants.
A fungal strain of Trichoderma having strong chitinolytic activity was isolated from field soil enriched with crabshell for several years. Based on 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence analysis and morphological characteristics, the fungus was identified as Trichoderma asperellum and named as Trichoderma asperellum T-5 (TaT-5). The fungus released lytic enzymes such as chitinase and ${\beta}$-1, 3-glucanse, and produced six antifungal substances in chitin broth medium. To demonstrate the protective effect of TaT-5 against damping-off in cucumber plant caused by Rhizoctonia solani, TaT-5 culture broth (TA), chitin medium (CM) and distilled water (DW) were applied to each pot at 10 days after sowing, respectively. Then, the homogenized hyphae of R. solani were infected to each pot at 1 week after TaT-5 inoculation. During experimental period, fresh weight of shoot and root in cucumber plant more increased at TA treatment compared to other treatments. PR-proteins (${\beta}$-1, 3-glucanase and chitinase) activities in cucumber leaves markedly increased at CM and DW treatments, but the activity slightly increased and then decreased at TA treatment at 3 days after infection of R. solani. The activity of PR-proteins activities in cucumber roots at all treatments decreased with time where the degree of decrement was more alleviated at TA treatment than CM and DW. These results suggest that the lytic enzymes (chitinase and ${\beta}$-1, 3-glucanse) and antifungal substances produced by TaT-5 can reduce the pathogenic attack by R. solani in cucumber plants.
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제안 방법
5 mM potassium ferricyanide). Chitinase activity was calculated by measuring O.D. at 420 nm, and these data were compared with data from a NAG standard curve for NAG concentration calculation. One unit of chitinase activity was defined as the amount of enzyme that liberated 1 μmol of NAG per h at 37℃.
Cucumber seedlings were grown at 27℃ in an artificially illuminated room with a 16 hrs (hours) photoperiod. Plants were harvested at 0, 1, 2, 3, 5, 7, 8, 9, 11 and 13 days after inoculation T. asperellum T-5 (TA). Each harvested Plants was then carefully washed in running tap water, and fresh weight of shoots and roots was measured.
The purposes of the research are to investigate lytic enzymes and antifungal substances released by T. asperellum T-5 (TaT-5) and to explain changes of PRproteins and lignification-related enzymes induced in cucumber plants in response to TaT-5 and R. solan causing damping off.
대상 데이터
Microorganism isolation Soils were collected from the field enriched with crabshell in Yeonggwang, Korea. Soils were serially diluted with sterile water until a rate of 10-5, inoculated on chitin agar medium containing colloidal chitin 0.
The isolated fungus was cultured in laminarin broth medium containing laminarin 0.5% ; Na2HPO4 0.2% ; KH2PO4 0.1% : NaCl 0.05% ; 0.1% ; NH4Cl 0.1% ; MgSO4·7H2O 0.05% ; CaCl2·2H2O 0.05% ; Yeast Extract 0.05% ; Agar 2% (pH 5) at 26℃ for 9 days on the 170 rpm shaking incubator. During shaking incubation, samples were taken at 1, 2, 3, 5, 7, 9 days.
이론/모형
, Japan) and it was eluted with stepwise gradient of water methanol (100:0, 50:50, 30:70, 0:100, v/v). Each fraction of the elute was concentrated using a evaporator, and the antifungal activity of each fraction against R. solani was conformed using the paper disk agar diffusion method. The 70% methanol and 100% methanol elute, which had strong antifungal activity, was purified using sephadex LH-20 column chromatography (25-100μ, SIGMA, Sweden) and it was eluted two times with methanol at a flow rate of 0.
성능/효과
(1999), the fungus was indentified as Trichoderma asperellum. Based on 5.8S rRNA and partial 18S and 28S rRNA genes, ITS1, ITS2 sequences, the fungus was identified as Trichoderma asperellum with 100% similarity to AJ230668.1 through BLASTN searching.
In conclusion, minor changes in levels of PR-proteins in TA may be responsible for the suppression of damping-off in cucumber associated with produced lytic enzymes and strong antibiotic compounds from TaT-5. Although the suppression activity of TaT-5 against damping off is not complete, the use of TaT-5 as a biocontrol agent may supplement or minimize that of synthetic chemicals.
, 2005). In this study, PR-proteins at the CM and the DW showed nearly no change in cucumber leaves until R. solani infection and then sharply increased. Interestingly, the increment of PR proteins in the TA was turned to decrement from 2 days after R.
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