| Diseases associated with Citrus tristeza virus (CTV) are a major bottleneck for Hawai'i's citrus growers. To increase citrus production, the incidence, distribution, and genetic diversity of Hawaiian CTV must first be characterized. From this data, control strategies can then be employed to manage this virus. In this study, a comprehensive survey of CTV was conducted and it was found that approximately three-quarters of Hawai'i's citrus trees were infected by CTV, and that no locations where citrus is currently grown are free of the virus. Genotyping using molecular markers and sequencing of coat protein and p23 genes of CTV revealed that the genetic diversity of Hawai'i's CTV population is also very high. Many trees appeared to be infected with multiple and/or recombinant virus strains, as well as previously unreported strains. To further characterize potentially novel strains of the virus, a dsRNA cloning strategy based on random (r)PCR and degenerate oligonucleotide primer (DOP)-PCR was developed that required sub-nanogram amounts of dsRNA template to construct cDNA libraries. Two new strains of CTV were completely sequenced using this strategy, one of which appeared to have arisen through modular recombination between two distantly-related strains of the virus. Given the robustness of Hawaii's CTV population, the development of CTV-resistant citrus via transgene silencing appears to be the most effective management option. Transgenes designed to counter this genetic diversity, as well as the three suppressors of gene silencing encoded in the CTV genome, were introduced into the Mexican lime (Citrus aurantifolia (Christm.) Swingle] genome. These transgenic plants are currently under evaluation for their ability to resist CTV infection. |
