Incidence, molecular characterization, genetic diversity and serological studies of pararetroviruses associated with Dahlia spp.
Almeyda, Christie Vanesa
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Two distinct caulimoviruses, Dahlia mosaic virus (DMV), and Dahlia common mosaic virus (DCMV), and an endogenous plant pararetroviral sequence (DvEPRS, formerly known as DMV-D10) were reported to be associated with dahlia mosaic, a serious disease affecting dahlia worldwide. Incidence studies carried out in 2010 demonstrated the predominance of DvEPRS over DCMV and DMV in cultivated dahlia (Dahlia variabilis). Molecular characterization of three new endogenous pararetroviral sequences (EPRSs) isolated from cultivated dahlias from Lithuania (D10-LT) and New Zealand (D10-NZ); and the wild species Dahlia rupicola from Mexico (D10-DR) was completed in this study. Genetic diversity studies were performed using a dataset of seven full-length EPRSs isolated from cultivated and wild Dahlia spp. Assessment of all open reading frames (ORFs) using phylogenomic and population genetics approaches showed that genetic diversity of EPRSs occurring in dahlia is very diverse. Phylogenetic analyses showed that EPRSs formed one clade, indicating a lack of clustering by geographical origin and no divergence due to source (cultivated vs. wild). Population genetic analyses found negative selection for all ORFs, with the reverse transcriptase region more variable than other ORFs. Recombination events were found and provided evolutionary evidence for genetic diversity. Promoter studies identified promoter elements of DMV, DCMV and DvEPRS and determinate their activity by transient expression of the â-glucuronidase (GUS) gene. Quantitative GUS assays showed that the promoters from DMV and DCMV resulted in higher levels of gene expression compared to that of Cauliflower mosaic virus 35S promoter in Nicotiana benthamiana leaf tissue, whereas low promoter activity was observed for DvEPRS. Serological studies developed an indirect plate-trapped antigen (PTA) Enzyme Linked Immunosorbent Assay (ELISA) method for the specific detection of DMV. The aphid transmission factor (ATF) gene was bacterially expressed and used as an antigen to produce antisera in rabbits. The anti-DMV-ATF serum detected the virus up to 1:10000 dilution of the antiserum using a bacterial cross-absorption procedure. Attempts to generate DMV- and DCMV-infectious clones were made to produce tools for investigating viral gene functions and virus-host interactions. This study contributes to an increased understanding of the incidence, genetic characterization, serology, and genetic diversity of caulimoviruses.