Vector-enabled metagenomics (VEM) approach to investigate the presence of potentially invasive geminiviruses in the south of Italy

Motivation Geminiviruses are monopartite or bipartite circular single-stranded DNA viruses, infecting different crops, such as cucurbits and tomatoes, in the Mediterranean area. Their economic impact is significant, and they are considered invasive pathogens due to frequent introduction of exotic species and their high level of recombination, which potentially generates new and resistance-breaking strains. To prevent and control the spread of new and invasive geminiviruses (NIGs), a vector-enabled metagenomics (VEM) approach has been used to identify the “circulating geminiviroma” in Southern Italy. Methods Insects belonging to the Bemisia spp. complex, leafhoppers, aphids, and mirids were collected using sweeping nets and/or entomological aspirators in the years 2020-2022 from open fields and plastic tunnels located in two eco-agrosystems, Sicily and Campania, from either crops or the spontaneous flora near the field borders, or from spontaneous grasses in the proximity of greenhouses. After DNA extraction, samples were organized in 15 different pools, based on the agroecosystem and date of collection. Circular DNAs were enriched using the Rolling Circle Amplification (RCA) and samples were sent to a specialized company for library construction and DNA sequencing with Illumina Novaseq 6000 (2x150bp). Raw data were checked for quality and adapter contamination with fastp (v. 0.21.0). Ribosomal sequences were removed with bbmap tool (v. 38.7). Reads were assembled into scaffolds using metaspades (v. 3.15.1); statistics on the assembly were performed with QUAST (v. 5.2.0). Redundancy reduction was performed with CAP3 (VersionDate 02/10/15). Only sequences longer than 500 bp were retained and used as input for diamond blastx (v. 2.0.15; diamond db updated at 13/05/2023). Results of blastx search were visualized with MEGAN (v. 6.25.9). In order to retrieve the full length genome of the viral species identified, clean reads were mapped against the viral reference genome downloaded from RefSeq database of NCBI using bowtie2 (v. 2.2.9). Results Except for one pool including myrids collected in Sicily in July 2021, where no geminiviruses were found, all the other 14 pools showed the presence of at least one plant viral species. Among the NIGs of particular interest, tomato leaf curl New Delhi virus (ToLCNDV) was present in almost all pools, tomato yellow leaf curl virus (TYLCV) was detected only in whiteflies collected in Sicily in October 2020 and July 2021 and sweet potato leaf curl virus (SPLCV) was recovered in whiteflies collected in Campania in July/November 2020. All three viruses have been already detected in Italy. Sequences related to other interesting viruses belonging to the family of Geminiviridae are under validation and insist on both eco-agrosystems. Other interesting viral sequences likely representing new members belonging to Geminiviridae and Genomoviridae families were also reconstructed. Validation through PCR and morphological insects’ identification are ongoing. We showed that the circulating virome analysis through vector-enhanced metagenomic (VEM) approach can be crucial in monitoring the presence of invasive viruses other than in monitoring the ongoing viral diseases transmitted by insects also in large areas, due to high insect mobility.