High Throughput Sequencing (HTS) – based identification and characterization of Geminiviridae family members in weeds associated with the tomato crop in Brazil

Abstract

The tomato (Solanum lycopersicum L.) crop has great economic and social importance for Brazil. Production of this vegetable is affected by many diseases, including those induced by Begomovirusspecies (family Geminiviridae). The Chapter 1 presents a review of these pathogens and the diseases they induce. In addition to tomato, begomoviruses have a wide range of hosts across different botanical families. Many weeds are natural hosts, playing an important role as reservoirs for this group of pathogens. Begomoviruses are transmitted by the Bemisia tabaci species complex, which are characterized by having a wide geographic distribution and being extremely polyphagous. These characteristics of the vectors increase the potential for mixed viral infections and for events of recombination and pseudo-recombination. Such events provide an increase in the variability of the viral populations, including the emergence of novel species and viral variants capable of overcoming resistance factors present in commercial varieties. In this context, monitoring viral diversity in tomato fields and in associated weeds is extremely important for effective management of these pathogens. Different molecular strategies have been employed for large-scale identification and characterization of begomoviruses, including High Throughput Sequencing (HTS) platforms. Herein, a broad metagenomics analysis of single-stranded, circular DNA viral species of the Geminiviridae family (especially from the genus Begomovirus) was conducted in weeds frequently occurring in tomato fields. Plants from the families Malvaceae, Euphorbiaceae, Amaranthaceae, Solanaceae, Asteraceae, Rubiaceae, Fabaceae, Lamiaceae, Cucurbitaceae, Convolvulaceae, and Brassicaceae were collected in field surveys carried out in all five Brazilian regions. In Chapter 2, 91 foliar samples of weeds exhibiting begomovirus-like symptoms (mosaic, mottled, chlorotic sectors, and dwarfism) were selected according to the year and collection site. Samples were collected in production areas and areas in the vicinity of tomato fields between 2003 and 2022. The samples were subjected to total DNA extraction, which was used as a template for performing Rolling Circle Amplification (RCA) assays. The selected samples were submitted to PCR tests with specific primers targeting conserved genomic regions of begomoviruses. The RCA pool was assembled with positive PCR samples and sent for sequencing on an Illumina Nova Seq 6000 platform aiming to obtain viral genomes. For botanical identification of the host species, the DNAs of a subgroup of weed plants were used as template in PCR assays using primers targeting the Rubisco and/or Maturase K barcoding genes. After the assembly and recovery of 100 contigs, 20 corresponded to 15 new species that are currently being characterized biologically and molecularly. In Chapter 3, the complete genome of a genetically distinct isolate representing a potential new species (denominated CE–076) was retrieved via HTS and validated via Sanger sequencing. This potential new virus displayed identity of 85.87% with tomato bright yellow mottle virus (ToBYMV) and it was detected infecting a novel Fabaceae host species – Bolusafra bituminosa. The information generated in the present dissertation can contribute to the establishment of management systems and generate information of interest on viral diversity for tomato breeding programs. Furthermore, the present work confirms the epidemiological importance the weed plants as reservoirs of viral species described infecting tomato as well as a potential role in the genetic evolution of populations of this group of pathogens in Brazil.