Journal of Korean Society of Forest Science (한국산림과학회지)

Korean Society of Forest Science (한국산림과학회)
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The Detection and Density Fluctuation of Mulberry Dwarf Phytoplasma using Nested-PCR and Competitive-PCR Methods

Nested-PCR법과 Competitive PCR법을 이용한 뽕나무 오갈병(MD) Phytoplasma의 검출과 밀도변화

  • Chae, Seungmin (Deartment of Forest Environmental Science, Chonbuk national University) ;
  • Lee, Sol (Deartment of Forest Environmental Science, Chonbuk national University) ;
  • Cha, Byeongjin (Department of Plant medicine, Chungbuk National University) ;
  • Lee, Hyokin (National Plant Quarantine Service) ;
  • Han, Sangsub (Deartment of Forest Environmental Science, Chonbuk national University)
  • 채승민 (전북대학교 산림환경과학과) ;
  • 이솔 (전북대학교 산림환경과학과) ;
  • 차병진 (충북대학교 식물의학전공) ;
  • 이혁인 (국립식물검역원) ;
  • 한상섭 (전북대학교 산림환경과학과)
  • Received : 2011.07.12
  • Accepted : 2011.08.12
  • Published : 2011.12.31
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Abstract

The detectable levels and population fluctuations of phytoplasmas infecting dwarf mulberry trees were investigated using nested-PCR and competitive-PCR methods. Samples of five different types were studied : A. petiole of a leaf that displays dwarf symptoms, B. petiole from apparently healthy leaf residing on a branch also supports a leaf with dwarf symptoms, C. the branch portion that supports a leaf with dwarf symptoms, D. the leaf petiole from healthy appearing leaves on branch with no dwarf symptoms, and branch portion of branch with no dwarf symptoms, E. the rootlets of trees with dwarf symptoms. These 5-parts were collected from each tree during June - April, once in every two months. The phytoplasma was detected from all parts of collected mulberry samples during all seasons using nested-PCR with AS-1/AS-2 primer pairs. The phytoplasma was detected until $10^4$ dilution using direct-PCR method, but it was detected until $10^{13}$ dilution by the nested-PCR method. The density of pytoplasma was found to be $7.94{\times}10^{18}-10^{12}copies/{\mu}L$ in mulberry trees. The density of phytoplasma was observed throughout the year in all samples of mulberry trees. The highest rates of phytoplasma was found in the samples B and C during the early growing season followed by the sample A and D during the dormant season. Samples C and E displayed the highest phytoplasma density followed sample D. The density of phytoplasma appeared stable during all the seasons for samples C and A. The result of the present study demonstrates the utility of nested-PCR and competitive-PCR for detection and determination of population fluctuations of phytoplasmas in plant tissues.

파이토플라스마에 감염된 뽕나무의 각 부위를 nested-PCR법과 경쟁 PCR법을 이용하여 파이토플라스마의 검출 범위 및 밀도 변화를 조사하였다. 파이토플라스마에 부분 감염된 뽕나무를 총 5부위(A. 빗자루병이나 오갈병 증상을 보인 잎의 엽병, B. 병징을 보인 잎이 있는 가지의 아래쪽 건전해 보이는 잎의 엽병, C. 병징을 보인 잎의 가지 부위, D. 병징을 보이지 않은 가지 쪽의 영병이나 가지, E. 잔뿌리)로 구분하였다. AS-1/AS-2 프라이머를 이용한 nested-PCR 결과, 파이토플라스마는 모든 시료에서 검출되었으며, 파이토플라스마의 검출범위는 P1/P7 프라이머를 이용한 direct-PCR 산물에서는 희석액이 $10^4$배까지 검출되었고, AS-1/AS-2 프라이머를 이용하여 nested-PCR를 수행한 결과, PCR 산물을 $10^{13}$배 희석한 시료까지 파이토플라스마가 검출되었다. 경쟁 PCR 결과, 뽕나무 파이토플라스마의 밀도는 $7.94{\times}10^{18}-10^{12}copies/{\mu}L$의 범위내에 존재하였으며, 년 중 파이토플라스마가 모든 시료에서 존재하였다. 초기생장시기에는 "B"부위와 "C"부위에서 파이토플라스마 밀도가 높게 나타났으나 "A"부위와 "D"부위에서는 이보다 낮게 나타났다. 휴면기에는 "C"부위와 "E"부위가 "D"부위보다 밀도가 높았다. 모든 시료 중 "C"부위와 "A"부위에서 년 중 파이토플라스마 밀도가 안전적으로 존재하였다. 본 연구 결과, nested-PCR 법과 경쟁 PCR법은 식물체내의 파이토플라스마의 정확한 검출과 밀도변화를 알아볼 수 있는 유용한 방법이다.

Keywords

References

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