First Report on the Occurrence of Grapevine leafroll-associated viruses 5 and 9 in Tunisian Grapevines.

Abstract

Grapevine leafroll disease is one of the most important diseases that occurs in cultivated grapevines in the world. So far, nine serologically distinct viruses of the family Closteroviridae have been isolated from diseased vines (3). A previous study (4) has shown that Grapevine leafroll-associated viruses (GLRaV) -1, -2, and -3 are present in Tunisian grapevines and GLRaV-3 is the predominant virus associated with leafroll disease. A survey was conducted in table grapes to identify other viruses associated with this disease. Samples of dormant canes were collected and screened by indirect Biotin Steptavidin ELISA with specific antibodies to GLRaV-5 (Bio-Rad, Sanofi, France) according to the manufacturer's instructions. Serological analysis revealed that nearly 47% of the samples were infected with GLRaV-5. To confirm GLRaV-5 identification and identify other leafroll viruses, vines with severe leafroll symptoms were collected and total RNA extracts were obtained from six samples and tested at Waite Diagnostics (University of Adelaide, Australia) by reverse transcription (RT)-PCR using primers for GLRaV-5 (2), LR5-1F 5'-CCCGTGATACAAGGTAGGACA-3' and LR5-1R 5'-CAGACTTCACCTCCTGTTAC-3' with a resulting amplicon size of 690 bp and primers for GLRaV-9, LR9F 5'-ACAGTGGTCGGCATAAGAAAAG-3' and LR9R 5'-ACACAAACATGCAGGCCAAAG-3' with a resulting amplicon size of 250 bp. Results showed that 1 of 6 and 5 of 6 of the samples were infected with GLRaV-5 and GLRaV-9, respectively, by RT-PCR and comparable results were obtained by ELISA. Amplicons were cloned and sequenced to confirm the identification of GLRaV-5 and GLRaV-9. The obtained sequences showed 99.1% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-5 (GenBank Accession No. AF233934) and 97.6% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-9 (GenBank Accession No. AY297819). The occurrence of GLRaV-9 has previously been reported in California and Australia (1). To our knowledge, this is the first report on the occurrence of GLRaV-5 and -9 in Tunisian grapevines. The widespread occurrence of GLRaV-5 and -9 is probably due either to the presence of their putative vectors, Planococcus ficus (Signoret) and Planococcus citri (Risso), or by propagation using infected local source material. Further studies are in progress to verify the implication of indigenous mealybugs in the spread of these viruses.