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

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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.