The single-stranded DNA, whitefly-transmitted begomoviruses are among the most serious emerging crop pathogens worldwide. To enhance current knowledge of begomovirus diversity, evolution, and biogeography, this project utilizes a novel approach of identifying begomoviruses directly from the Bemisia tabaci whitefly vector. This approach exploits the natural ability of whiteflies to concentrate viruses from the many plants they feed upon, and leverages the capability of metagenomic tools for discovering new viruses. Whitefly samples will be collected from tomato and squash fields from eight countries on six continents. Metagenomic sequencing will yield numerous complete begomovirus genome sequences, which will be used to improve our knowledge of begomovirus phylogeny and evolution. Deep sequencing of the begomovirus core coat protein region will enable direct comparisons of begomovirus diversity between crops and geographic locations.
This global vector-based survey will substantially increase our understanding of begomovirus diversity, recombination, biogeography, and emergence. Since begomoviruses are dangerous plant pathogens that pose a significant threat to global food biosecurity, this project has significant implications for agriculture. A graduate student and a postdoctoral researcher will be trained in cutting-edge molecular biology and bioinformatics techniques through this project. In addition, a Girl Scout "Virus Hunter" patch program will be developed to introduce young women to research in the detection and control of emerging viruses.
Whiteflies (Bemisia tabaci MEAM1) have the ability to transmit a large number of plant viruses and are one of the most detrimental pests in agriculture. Whiteflies have been associated with a large number of plant viruses but very few other types of viruses. The majority of the viruses known to be transmitted are DNA plant viruses in the genus Begomovirus (Geminiviridae) and a few RNA plant viruses. 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 viruses present in these whiteflies. Our results indicate that there is evidence for many more viruses present in this whitefly than previously known. Metagenomic analysis of viral DNA extracted from the whiteflies found a large number of sequences with homology to a range of begomoviruses. In contrast, this same approach identified sequences similar to RNA viruses which appeared to be related to very divergent virus groups, and include many sequences that suggest the presence of new undescribed viruses. We followed up on sequences that indicated the presence of a whitefly-transmitted RNA plant virus, Cowpea mild mottle virus (CpMMV Florida) (genus Carlavirus). Using primers designed from the metagenomic sequences, we were able to identify the presence of CpMMV Florida in native and cultivated plant species growing near the original field site of whitefly collection. This is the first recognition of this virus in North America. CpMMV Florida is most closely related to a virus with a very limited geographic range in northern Argentina. We were able to transmit this virus from plant to plant by whiteflies and by mechanical inoculation. We established cultures of 3 isolates obtained by selection of single local lesions and all 3 were sequenced and determined to be identical. In a broader sense, the results of our work indicate that the current classification of the genus Carlavirus needs to be reevaluated. While there are a number of coat protein sequences of what is described as CPMMV there are few full length sequences. Our sequences will help resolve some of the confusion and may lead to a division of this virus species into what most likely will be multiple species. Our results show that metagenomics is an excellent approach to identify unknown viruses. Our metagenomic analyses indicate that there is a wide array of viruses present in the whiteflies we examined. Most of the DNA plant virus sequences present in the whitefly were dominated by begomoviruses. However, the data suggest that there was a much greater diversity of RNA viruses present in this whitefly. While we were able to validate the presence of one virus, however more work needs to be done to identify the other viruses.
