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한국잔디에 발생한 달라스팟 병의 주요 원인균인 Sclerotinia homoeocarpa의 동정 및 특성
박대섭,김경덕,염주립,오병석,박병선,Park Dae-Sup,Kim Kyong-Duck,Yeom, Su-Rip,Oh Byung-Seog,Park Byoung-Sun 한국잔디학회 2005 한국잔디학회지 Vol.19 No.2
위에서 골프장의 안양 중지 이병 잎에서 분리한 병원균인 달라스팟 유사 병원균을 분리 동정하였다. 2000년도 국내에 처음 보고된 이후 현재까지 뚜렷하게 진전된 연구가 보고 되지 않아 향후 사전 예방과 발생시 방제 법을 강구하기 위함과 아울러 코스관리자의 면밀한 관심을 유도하기 위하여 병원균 분리, 동정 및 특성을 한지형 잔디(Agyostis palustris Huds. cv Penncross)에서 발생하는 달라스팟 병원균과 비교 실험을 실시하였다. 1. 기존에 알려진 것처럼 Sclerotinia homoeocarpa 에서 보이는 온도에 대한 민감도를 새로운 병원성 Scz1 균주에서도 볼 수있어 특정온도에서의 생장억제는 안양 중지 달라스팟 병의 발병하는 시기뿐만 아니라 병방제관리에 도움이 될 것으로 생각된다. 2. 병원균 균사 특성에서 전자현미경사진으로는 구별하기 어려웠으나, PDA배지상에서의 Scz1와 Scz2 색상은 동일한 반면 Scb1균사색상과는 차이가 남으로 향후 간단하게 균사색상으로 식별이 용이 할 것이다. 3. 실험실상에서 병원균의 약제 민감도 측정결과는 이프로디온과 프로피코나졸 약제가 우수하였다. 4. 병 접종 실험을 통하여 분리된Sczl 균주가 안양 중지에 대한 기주 특이성을 보임으로 조이시아 달라스팟의 주요 병원균으로 밝혀졌다. 5. Scz1의 병원성은 안양 중지에 대한 기주 특이성이 높은 것으로 밝혀졌다. A fungal isolate was newly collected from Zoysiagrass 'Anyang-Joongji' in small circular patches on a fairway ofa golf course in Korea, which seriously occurred during the early summer period of 2005. The isolate presented on PDAmedium, named Scz1, was closely identical to Sclerotinia homoeocarpa, a casual fungus of dollar spot disease, in cool season turf grasses such as creeping bentgrass. Hereby, this study was accomplished to characterize the isolate and compare it with the fungus, named Scb1, isolated from dollar spot-infected creeping bentgrass (Agrostis palustris Huds. cv Penncross). On PDAmedium, individual mycelial appearance of three isolates was very similar except for the pigment. Mycelial pigments of Scz1 and Scz2 (another analogous isolate collected) were light pinkish on the reverse side of PDA medium but that of Scb1 was dark brownish. In a microscopic study, three isolates were barely distinguishable in the appearance of mycelia. As expected, in the temperaturesensitivity assay, all pathogens were very delicate to $32^{circ}C$ above but not to $30^{circ}C$ below, in which was explained to be one of typical characteristics in S. homoeocarpa. In an artificial inoculation assay, disease symptoms including leaf spots in Zoysiagrass were appeared within 6-7 days after inoculation through the hand inoculation method with the isolate-infested soil. Then the fungus was re-identified from the infected leaf tissues. Interestingly, inoculation of isolate Scz1 gave rise to distinct symptoms in only Zoysiagrass but not in creeping bentgrass 'Penncross' and Kentucky bluegrass 'Midnight'. The observation might be involved in host specific pathogenecity of S. homoeocarpa Scz1 to Zoysiagrass. In a chemical sensitivity assay for the isolate, Scz1, showed a high mycelial inhibition against two fungicides, iprodione and propiconazole. All results described above suggest that S. homoeocarpa Scz1 is a primary pathogen of Zoysia dollar spot disease.
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유전자총과 아그로박테리움을 이용한 여러 가지 한국 잔디류의 형질전환체계 확립
김종보,김경덕,박대섭,Kim Jong-Bo,Kim Kyong-Duck,Park Dae-Sup 한국잔디학회 2004 한국잔디학회지 Vol.18 No.3
다양한 Zoysiagrass 4가지 품종들을 식물재료로 사용하여 Agrobacterium만 이용한 방법 그리고 particle bombardment로 배발생캘러스에 상처를 낸 후, Agrobacterium으로 공동배양 시키는 2가지 다른 형질전환 방법을 비교하였다. 예비실험에서 일반적으로 형질전환에 널리 사용되는 kanamycin과 PPT(phospinitricin)의 적적선발농도에 대해서 실험하였는데, kanamycin의 경우 300mg/l 그리고 PPT의 경위 50mg/l의 농도에서 가장 효과적인 선발 효율을 나타내었다. Agrobacterium을 이용한 형질전환은 Agrobacterium을 2일간 배양시킨 다음, 박테리아 농도를 O.D 600nm=1.0-1.2로 맞추고, 배발생캘러스를 30분간 간염 시키는 방법이 효과적이었는데, particle bombardment를 이용하여 캘러스에 상처를 유발시킨 후, Agrobacterium으로 감염시키면 3배 이상 높은 형질전환 수율을 획득할 수 있었다. 이상의 결과는 한국잔디 형질전환에 있어서 particle bombardment과 Agrobacterium을 병행하여 실시한 최초의 보고이고, 이러한 시스템을 기반으로 하여 향후 한국잔디를 포함하여 다른 난지형 및 한지형 잔디의 품종개량에 널리 이용되리라 생각된다. In this report, several factors such as infection time and concentration of bacterial suspension, influencing on transient gene expression in Agrobacterium-mediated transformation were evaluated. An appropriate concentration (O.D 600nm = 1.0-1.2) of bateria and 30 min of infection time showed a higher level of GUS expression. To improve transformation efficiency (TE), friable embryogenic calli (FEC) were bombarded by tungsten particles without plasmid DNA, and then co-cultivated with A. tumefaciens LBA4404 which contains pTOK233 super binary vector, carrying neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (hpt) and$\beta-glucuronidase$ (GUS) genes. Three days after co-cultivation with A. tumefaciens and particle bombardment, FEC cultures were transferred to the selection medium (SM: MS medium supplemented with BA 1mg/l, hygromycin 100mg/l, cefotaxime 250 mg/l and vancomycin 200mg/l). They were cultured for 2 weeks and then transferred to the second SM containing hygromycin 50mg/l, cefotaxime 200 mg/l and vancomycin 100mg/l. Later, stable GUS expression was detected 4 to 6 weeks after transfer to the SM. Further, TE from Agrobacterium-mediated transformation after particle bombardment increased to about 3-folds compared with Agrobacterium-mediated transformation without particle bombardment. In the present study, we established an efficient transformation protocol of zoysiagrass by using A. tumefaciens in the combination with particle bombardment for the first time.
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한국잔디에 발생한 달라스팟 병의 주요 원인균인 Sclerotinia homoeocarpa의 동정 및 특성
박대섭(Dae-Sup Park),김경덕(Kyong-Duck Kim),염주립(Ju-Rip Yeom),오병석(Byung-Seog Oh),박병선(Byoung-Sun Park) 한국잡초학회·한국잔디학회 2005 Weed & Turfgrass Science Vol.19 No.2
A fungal isolate was newly collected from Zoysiagrass 'Anyang-Joongji' in small circular patches on a fairway ofa golf course in Korea, which seriously occurred during the early summer period of 2005. The isolate presented on PDAmedium, named Scz1, was closely identical to Sclerotinia homoeocarpa, a casual fungus of dollar spot disease, in cool season turf grasses such as creeping bentgrass. Hereby, this study was accomplished to characterize the isolate and compare it with the fungus, named Scb1, isolated from dollar spot-infected creeping bentgrass (Agrostis palustris Huds. cv Penncross). On PDAmedium, individual mycelial appearance of three isolates was very similar except for the pigment. Mycelial pigments of Scz1 and Scz2 (another analogous isolate collected) were light pinkish on the reverse side of PDA medium but that of Scb1 was dark brownish. In a microscopic study, three isolates were barely distinguishable in the appearance of mycelia. As expected, in the temperaturesensitivity assay, all pathogens were very delicate to 32℃ above but not to 30℃ below, in which was explained to be one of typical characteristics in S. homoeocarpa. In an artificial inoculation assay, disease symptoms including leaf spots in Zoysiagrass were appeared within 6-7 days after inoculation through the hand inoculation method with the isolate-infested soil. Then the fungus was re-identified from the infected leaf tissues. Interestingly, inoculation of isolate Scz1 gave rise to distinct symptoms in only Zoysiagrass but not in creeping bentgrass 'Penncross' and Kentucky bluegrass 'Midnight'. The observation might be involved in host specific pathogenecity of S. homoeocarpa Scz1 to Zoysiagrass. In a chemical sensitivity assay for the isolate, Scz1, showed a high mycelial inhibition against two fungicides, iprodione and propiconazole. All results described above suggest that S. homoeocarpa Scz1 is a primary pathogen of Zoysia dollar spot disease.
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유전자총과 아그로박테리움을 이용한 여러 가지 한국 잔디류의 형질전환체계 확립
김종보(Jong-Bo Kim),김경덕(Kyong-Duck Kim),박대섭(Dae-Sup Park) 한국잡초학회·한국잔디학회 2004 Weed & Turfgrass Science Vol.18 No.3
In this report, several factors such as infection time and concentration of bacterial suspension, influencing on transient gene expression in Agrobacterium-mediated transformation were evaluated. An appropriate concentration (O.D600nm = 1.0-1.2) of bateria and 30 min of infection time showed a higher level of GUS expression. To improve transformation efficiency (TE), friable embryogenic calli (FEC) were bombarded by tungsten particles without plasmid DNA, and then co-cultivated with A tumefaciens LBA4404 which contains pTOK233 super binary vector, carrying neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (hpt) and ß-glucuronidase (GUS) genes. Three days after co-cultivation with A tumefaciens and particle bombardment, FEC cultures were transferred to the selection medium (SM' MS medium supplemented with BA l mg/l, hygromycin 100 mg/l, cefotaxime 250 mg/l and vancomycin 200 mg/l). They were cultured for 2 weeks and then transferred to the second SM containing hygromycin 5O mg/l, cefotaxime 200 mg/I and vancomycin 100 mg/l. Later, stable GUS expression was detected 4 to 6 weeks after transfer to the SM. Further, TE from Agrobacterium-mediated transformation after particle bombardment increased to about 3-folds compared with Agrobacterium-mediated transformation without particle bombardment. In the present study, we established an efficient transformation protocol of zoysiagrass by using A tumefaciens in the combination with particle bombardment for the first time.
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