Japanese Journal of Phytopathology
Online ISSN : 1882-0484
Print ISSN : 0031-9473
ISSN-L : 0031-9473
Selection of PCR Primers and a Simple Extraction Method for Detection of Hop Stunt Viroid-plum in Plum by Reverse Transcription-Polymerase Chain Reaction
Nario KUSANOKatsumi SHIMOMURA
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1997 Volume 63 Issue 2 Pages 119-123

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Abstract

Several conditions were examined to optimize the detection of hop stunt viroid-plum, a causal pathogen of plum dapple fruit disease, by reverse transcription-polymerase chain reaction (RT-PCR). The best primer pair of the six examined was HSVc-1 (5'-GGCTCCTTTCTCAGGTAAG-3')/HSVs-2 (5'-CCGGGGCAACTCTTCTCAGAATCCA-3'). By using this primer pair, the RT-PCR assay was about 10, 000 times more sensitive than two-dimensional polyacrylamide gel electrophoresis, judging from a dilution end point experiment. Treatment of CF-11 cellulose column chromatography before RT-PCR was effective in removing inhibitors present in plum sap extracts. SepaGene RV-R was satisfactory as a simple extraction method to extract the viroid from barks, roots and leaves of plum for RT-PCR assays. The extracts from barks and roots gave better PCR amplification than leaf extracts. The direct nucleotide sequencing of PCR-amplified cDNAs of the viroid isolated from dapple fruit plum (cvs. Ooishiwasesumomo and Soldam) revealed that the viroid was identical to HSVd-plum reported in plum (cv. Taiyo) in Yamanashi Prefecture.

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