Grapevine vitivirus A eradication in Vitis vinifera explants by antiviral drugs and thermotherapy

Grapevine shoot cultures infected by Grapevine vitivirus A (GVA) were grown on Quorin-Lepoivre basic medium and submitted to in vitro chemotherapy and thermotherapy sanitation techniques. Ribavirin (Rb) at 20gml(-1), dihydroxypropyladenine (DHPA) at 60gml(-1) and their combination (RbDH) were added to the proliferating medium for three subsequent subcultures of 30 days each. Phytotoxicity was observed on drug-treated plantlets, which displayed a high percentage of mortality for each drug at doses higher than those aforementioned. Sequential ELISA were performed at the end of each subculture and ELISA-negative explants were submitted to RT-PCR. ELISA showed no antiviral activity following DHPA administration. Rb and RbDH treatment produced ELISA-negative explants which were assayed by RT-PCR and nested PCR. Biomolecular results showed no virus eradication in Rb treated explants but RbDH administration generated a percentage (40.0%) of GVA-free plantlets that permitted restoration of a new healthy generation of explants. Sixty percent (60%) of GVA eradication as confirmed by RT-PCR was obtained by in vitro thermotherapy at 36 degrees C for 57 days.