Current knowledge of plant virus diversity is heavily biased towards agents of visible and economically important diseases, and we know little of viruses that have not yet appeared in epidemic proportions in crop plants. The 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 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.
The whitefly-transmitted begomoviruses (Geminiviridae) are one of the most damaging and emergent plant pathogens worldwide. This study significantly expanded our knowledge regarding begomovirus diversity, evolution, and biogeography through the application of a novel approach examining begomoviruses directly from their whitefly vector (Bemisia tabaci). Whiteflies feed on a very wide range of plants and are highly mobile, being able to fly short distances and capable of traveling up to a few kilometers when assisted by the wind. Our approach, known as vector-enabled metagenomics, leveraged the sampling ability of whiteflies with a sequence-independent approach (metagenomics) that allowed us to recover novel begomoviruses. In addition, we employed a sequence-dependent approach (PCR) that allowed us to examine the distribution of genetic variants and directly compare different sites and crops. Vector-enabled metagenomics, as initiated through this work, has been transformative for the field of plant virology by enabling rapid and efficient description of the begomoviruses circulating in whiteflies within a given region. This vector-based approach effectively integrates over space and time, and provides advantages over standard methods by enabling the identification of viruses infecting native vegetation, newly emerging viruses that are not yet widespread in crops, and viruses that have more mutualistic interactions with their host. This project analyzed begomovirus diversity in whiteflies collected from crops (e.g., tomato, squash) and uncultivated vegetation (weeds) from Brazil, Guatemala, Israel, Puerto Rico, Spain, and the continental United States (California and Florida). Based on a PCR assay targeting the capsid protein (CP) gene, a total of 177 distinct begomovirus variants were detected. Whiteflies collected from a squash crop in Guatemala exhibited the highest diversity with 23 distinct variants. Although similar viruses were found in several locations, the distribution of specific genetic variants was driven by a given location rather than specific plant hosts where whiteflies were collected. The metagenomic analysis revealed the presence of several monopartite begomoviruses that the PCR assay failed to detect, including Sweet potato leaf curl virus (Spain and Puerto Rico), Hollyhock leaf crumple virus (Israel), and Cotton leaf curl Gezira virus (Israel), stressing the importance of including sequence-independent surveys to capture total begomovirus diversity. Novel bipartite begomovirus species were detected in samples from Guatemala and Puerto Rico and a novel monopartite begomovirus similar to Tomato yellow leaf curl virus (TYLCV) was detected in samples from Spain. In addition, this analysis allowed the detection of begomovirus-associated satellite DNAs, resulting in the biogeographical expansion of some of these molecules and showing that satellite DNAs are more widespread in the New World than previously recognized. In total, 81 circular genomic components were completely sequenced, including full begomovirus genomes (DNA-A and B), partial begomovirus genomes (DNA-A), and satellite molecules. This project provided training for one postdoctoral researcher and two undergraduate students. Numerous outreach activities were performed during the course of this project to introduce the concept of whiteflies and other insect vectors as "flying syringes" that can sample viruses from many hosts over space and time. Finally, throughout the course of this project, we had the opportunity to expand upon the vector-enabled metagenomics approach and utilize this method to examine other insect vectors (e.g., mosquitoes) and other virus types (e.g., RNA viruses). These studies demonstrated the broader applicability of the vector-enabled metagenomics approach. This method was also adapted to study dragonflies, which are top insect predators. Predator-enabled metagenomics on dragonflies integrates over the many flying insects that dragonflies feed upon and was shown to be capable of discovering diverse virus types. In conclusion, this global survey provided unparalleled insight regarding the diversity, evolution, and biogeography of begomoviruses. In addition to significantly increasing our awareness of begomovirus diversity, the application of vector-enabled metagenomics to whiteflies and other insect vectors can provide an effective molecular surveillance system capable of recognizing introduced and emerging viruses of great societal importance for the agriculture and public health sectors.
