Virus elimination from Actinidia germplasm

Abstract

Kiwifruit (genus Actinidia) is an important horticultural crop to New Zealand. Since 2003 several viruses have been isolated from Actinidia germplasm. Because the germplasm is difficult to replace it is vital that viruses are eliminated from the infected material. The anti-viral effect of thermotherapy and chemotherapy was tested on a selection of viruses that infect Actinidia species (Actinidia virus A (AcVA), Actinidia virus B (AcVB), Alfalfa mosaic virus (AMV), Apple stem grooving virus (ASGV), Actinidia virus X (AVX), Citrus leaf blotch-like virus (CLBV-a), and Cucumber mosaic virus CMV). Also, the phylogenetic relationship of AMV isolated from Actinidia was compared to other extant isolates. It was found that Actinidia chinensis and Actinidia deliciosa shoot tip cultures did not remain infected with ASGV, AVX, CLBV-a, and CMV, indicating that tissue culturing may have eliminated these viruses or that they do not persist in these species. Exposure of A. deliciosa to a range of elevated temperatures (30-40°C) showed that the cultures do not survive at 37.5°C or higher. Application of a temperature cycling regime of 25°C for 8 hours and 35°C for 16 hours improved the tolerance of A. chinensis and A. deliciosa to incubation at an elevated temperature. When the temperature cycling regime was applied to AcVA, AcVB, AMV, ASGV, AVX, CLBV-a, and CMV in Nicotiana occidentalis shoot tip cultures, the treatment was able to reduce the titre of all the viruses studied. The effects of anti-viral compounds (acyclovir, amantadine hydrochloride, 5-azacytidine, oseltamivir, ribavirin, 2-thiouracil, and vidarabine) against AMV, ASGV, AVX, CLBV-a, and CMV in N. occidentalis shoot tip cultures cultures was determined. Only ribavirin (20mg/L) and ribavirin (10mg/L) combined with quercetin (10mg/L) had a significant effect against ASGV and CLBV-a. These treatments were not phytotoxic to Actinidia and, along with thermotherapy, are promising candidates for further development of virus elimination protocols in Actinidia. Phylogenetic analysis of RNA 3 and the coat protein sequences of AMV isolated from three Actinidia species showed the presence of two distinct sequence variants that are dissimilar to other AMV isolates so far identified in New Zealand; these isolates likely represent two separate introductions, one or both of which may represent an Actinidia strain.