Genomovirus Genomes Recovered from Echinothrips americanus Sampled in Florida, USA.

Four genomovirus genomes were recovered from thrips (Echinothrips americanus) collected in Florida, USA. These represent four new species which are members of the Gemycircularvirus (n = 2), Gemyduguivirus (n = 1), and Gemykibivirus (n = 1) genera. This is the first record, to our knowledge, of genomoviruses associated with a phytophagous insect.

RNA viral metagenome of whiteflies leads to the discovery and characterization of a whitefly-transmitted carlavirus in North America.

Whiteflies from the Bemisia tabaci species complex have the ability to transmit a large number of plant viruses and are some of the most detrimental pests in agriculture. Although whiteflies are known to transmit both DNA and RNA viruses, most of the diversity has been recorded for the former, specifically for the Begomovirus genus. This study investigated the total diversity of DNA and RNA viruses found in whiteflies collected from a single site in Florida to evaluate if there are additional, previously undetected viral types within the B. tabaci vector. Metagenomic analysis of viral DNA extracted from the whiteflies only resulted in the detection of begomoviruses. In contrast, whiteflies contained sequences similar to RNA viruses from divergent groups, with a diversity that extends beyond currently described viruses. The metagenomic analysis of whiteflies also led to the first report of a whitefly-transmitted RNA virus similar to Cowpea mild mottle virus (CpMMV Florida) (genus Carlavirus) in North America. Further investigation resulted in the detection of CpMMV Florida in native and cultivated plants growing near the original field site of whitefly collection and determination of its experimental host range. Analysis of complete CpMMV Florida genomes recovered from whiteflies and plants suggests that the current classification criteria for carlaviruses need to be reevaluated. Overall, metagenomic analysis supports that DNA plant viruses carried by B. tabaci are dominated by begomoviruses, whereas significantly less is known about RNA viruses present in this damaging insect vector.

Inoculation of plants with begomoviruses by particle bombardment without cloning: Using rolling circle amplification of total DNA from infected plants and whiteflies.

A new system for inoculation of plants with begomoviral DNA without cloning or the use insect vectors is described. Total DNA extracted from begomovirus-infected plants was amplified by rolling circle amplification (RCA) using the bacteriophage phi29 DNA polymerase, and inoculated to plants by particle bombardment. Infection rates of up to 100% were obtained using this technique. This technique successfully inoculated all the begomoviruses evaluated: five bipartite (Bean golden yellow mosaic virus, Cabbage leaf curl virus, Squash leaf curl virus, Tomato mottle virus, Watermelon chlorotic stunt virus) as well as one monopartite (Tomato yellow leaf curl virus). The success of the technique was not dependent upon plant species. Four species from three plant families [Phaseolus vulgaris (bean), Solanum lycopersicum (tomato), Cucurbita pepo (squash), and Citrullus lanatus (watermelon)], could all be inoculated by this technique. The success of the method was not dependent upon either the type or the age of the source of virus. Infectious DNA was obtained successfully from fresh, freeze-dried or desiccated plant material, from squashes of plant leaves on FTA cards, as well as from the insect vector. Plant material collected and dried as long as 25 years ago yielded infectious DNA by this method. In summary, this method can be used to obtain infectious DNA of single-stranded circular DNA viruses that can be activated for purposes of completing Koch's postulates, for preservation of pure virus cultures, and for many other applications where infectious DNA is required.