DETECTION OF GRAPEVINE LEAFROLL-ASSOCIATED CLOSTEROVIRUS-III BY MOLECULAR HYBRIDIZATION

Highly purified double-stranded (ds) RNA was obtained from cortical sc rapings of mature canes of vines infected by grapevine leafroll-associ ated closterovirus III (GLRaV III) using phenol-chloroform extraction, chromatography on CF-II cellulose minicolumns and enzymatic digestion . Complementary (c) DNA fragments of various lengths, obtained by rand om priming denatured dsRNA templates, were cloned into the plasmid pUC -18 at the SmaI site in Escherichia coli strain DH5 alpha. Two P-32-la belled cDNA clones denoted p16ds(c. 1100 bp) and p23ds (c. 1500 bp) we re successfully used as probes for detecting GLRaV III sequences in gr apevine extracts from leaves and petioles, or cortical tissues. Probe p23ds was virus-specific and did not hybridize with total RNA from hea lthy controls, or from vines infected by grapevine leafroll-associated closterovirus I (GLRaV I), or with genomic RNA from purified grapevin e closterovirus A (GVA) and B (GVB). A riboprobe (pGEM23ds) transcribe d from p23ds in transcription vector pGEM3zf specifically recognized G LRaV III sequences, but not GLRaV I or GVA sequences, in extracts from differently infected vines. Moreover, in Northern blots, the same pro be hybridized also with smaller dsRNA components, which may be replica tive forms of subgenomic RNAs.