Sensitive PCR-based Detection of Apple Chlorotic Leaf Spot Virus Heterogeneous in Apple Trees

• NAKAHARA Kenji S.

  Research Faculty of Agriculture, Hokkaido University

• YOSHIDA Kouji

  Apple Research Station, National Institute of Fruit Tree Science, National Agriculture and Food Research Organization (NARO)

• SUZAKI Kouichi

  Grape and Persimmon Research Station, National Institute of Fruit Tree Science, NARO

• YOSHIKAWA Nobuyuki

  The United Graduate School of Agricultural Sciences, Iwate University Plant Pathology Laboratory, Faculty of Agriculture, Iwate University

• ITO Tsutae

  Apple Research Station, National Institute of Fruit Tree Science, National Agriculture and Food Research Organization (NARO)

抄録

This paper presents a useful process for the detection of Apple chlorotic leaf spot virus (ACLSV) in apple trees. The 3′-terminal 1.8-kb genomic cDNAs of 15 ACLSV isolates, of which 11 induce a decline (of varying severity) in the condition of inoculated Malus prunifolia var. ringo rootstock and four do not, were amplified by reverse transcription (RT) coupled with polymerase chain reaction (PCR). Single-strand conformation polymorphism analysis of the cDNAs revealed that most of the isolates inducing the decline were composed of at least two to four sequence variants per apple tree, whereas isolates inducing no decline were occupied with a major type sequence. Direct sequencing of the cDNAs showed heterogeneity in the nucleotide sequence of the analyzed region; most of the variation in these positions appeared to specify the same amino acids upon translation of the 50K protein and capsid protein (CP). To detect all isolates, degenerate primers were designed with consideration of the sequence varieties of the viral genomes. RT-nested PCR and its improved methods have a 10⁴-fold higher sensitivity than conventional RT-PCR, and consistently amplify ACLSV genomic cDNA in RNA extracts from apple leaf and bark during growing and dormant seasons, respectively.

収録刊行物

• Japan Agricultural Research Quarterly: JARQ

  Japan Agricultural Research Quarterly: JARQ 45 (4), 411-421, 2011

  国立研究開発法人　国際農林水産業研究センター

キーワード

• Topworking disease

詳細情報

• CRID: 1390001204701332864
• NII論文ID: 130002147955
• DOI: 10.6090/jarq.45.411
• ISSN: 21858896
• ISSN: 00213551
• 本文言語コード: en
• データソース種別:
  • JaLC
  • Crossref
  • CiNii Articles
  • KAKEN
• 抄録ライセンスフラグ: 使用不可