Detection of grapevine leafroll‐associated closterovirus III by molecular hybridization

Highly purified double-stranded (ds) RNA was obtained from cortical scrapings of mature canes of vines infected by grapevine leafroll-associated closterovirus III (GLRaV III) using phenol-chloroform extraction, chromatography on CF-11 cellulose minicolumns and enzymatic digestion. Complementary (c) DNA fragments of various lengths, obtained by random priming denatured dsRNA templates, were cloned into the plasmid pUC-18 at the Smal site in Escherichia coli strain DH5α. Two 32P-labelled cDNA clones denoted p16ds (c. 1100 bp) and p23ds (c. 1500 bp) were successfully used as probes for detecting GLRaV III sequences in grapevine extracts from leaves and petioles, or cortical tissues. Probe p23ds was virus-specific and did not hybridize with total RNA from healthy controls, or from vines infected by grapevine leafroll-associated closterovirus I (GLRaV I), or with genomic RNA from purified grapevine closterovirus A (GVA) and B (GVB). A riboprobe (pGEM23ds) transcribed from p23ds in transcription vector pGEM3zf specifically recognized GLRaV III sequences, but not GLRaV I or GVA sequences, in extracts from differently infected vines. Moreover, in Northern blots, the same probe hybridized also with smaller dsRNA components, which may be replicative forms of subgenomic RNAs.