NATURAL SPREAD AND MOLECULAR ANALYSIS OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUS-3 IN AUSTRALIA

Natural spread of grapevine leafroll disease was observed in a Pinot N oir clonal evaluation trial in South Australia. The trial consisted of 13 clones with the spread apparently initiated from 3 leafroll-infect ed clones: Antav 543, Geisenheim 20, and Bourgogne H199A. The occurren ce of grapevine leafroll-associated virus 3 (GLRaV-3) was suspected. A ll 104 vines in the trial were tested by enzyme-linked immunosorbent a ssay using antibody to GLRaV-3 and by slot blot hybridization analysis using double-stranded RNA as target and labeled GLRaV-3-specific cDNA as probe. Both tests linked GLRaV-3 with the disease spread and detec ted the infection prior to the onset of symptoms. A cDNA clone from an Italian isolate of GLRaV-3 hybridized in Northern blots with three ma jor dsRNAs of 19.5, 1.9, and 0.9 kbp extracted from leafroll-infected vines. The cloned cDNA insert of approximately 1 kbp was sequenced, an d a set of primers designed based on the sequence was used to obtain a corresponding polymerase chain reaction product from the Antav 543 is olate grown in Australia. The nucleotide sequence of the cDNA clones f rom the two isolates of GLRaV-3 showed 99.5% identity and contained an open reading frame (ORF) encoding a putative protein with a molecular mass of 20.4 kDa with no significant homology to known protein sequen ces. This ORF was mapped near the 3'-end of the plus strand viral geno me and had a 3'-untranslated AU-rich region of approximately 347 nucle otide residues.