The multi-actor approach methodology has reached 152 officially affiliated stakeholders across different regions. Since the project’s start, 409 participants attended 7 workshops in Germany, France, Spain, and Belgium; 299 students were trained, and 14 practice abstracts were published. Together with WP5, a questionnaire on whitefly control was distributed to 208 growers, aided by NKBs and national partners. The protocols for full-length sequencing of ToBRFV and ToLCNV with Oxford Nanopore Technology (ONT) were successfully implemented. A pipeline for accurate analysis of full-length virus sequences was implemented on the Genome Detective web platform by EMWEB. Samples of crops and weed hosts infected with DNA begomoviruses (Solanaceae and Cucurbitaceae) or RNA tobamoviruses (Solanaceae) were collected and sequenced by KUL to study virus diversity, distribution, and factors driving virus evolution. Wild tomato accessions asymptomatic and negative for ToBRFV were crossed with the susceptible variety Moneymaker. Tomato plants with high level of resistance to TYLCV were also asymptomatic to ToLCNDV. Tomato plants with high levels of resistance to betasatellites were developed. To understand the mechanism of resistance to whiteflies, transcriptomes of resistant cucumber lines are under study. To understand interactions between begomovirus deltasatellites and whiteflies, ten mutants of the deltasatellite SPLCD1 were designed. Infectious ToLCNDV clones were developed to test for resistance breakers. An infectious ToBRFV clone was developed. Transcriptome of early defence response to ToBRFV was generated. The seed-borne nature of ToLCNDV and ToBRFV was determined. Both viruses were detected in seeds from infected plants, but only ToBRFV was seed-transmissible. The role of alternative hosts in disease spread was assessed, revealing a limited number of wild hosts which allows effective disease control. Local whiteflies were better transmitters of ToLCNDV than invasive species, aligning with the co-adaptation hypothesis. Host jump by ToLCNDV was linked to viral proteins AC2/AC3 on DNA-A and the DNA-B component. Higher temperatures increased ToLCNDV transmission efficiency. Mixed infections (ToLCNDV + WMV; ToBRFV + PepMV) exacerbated plant damage in tomatoes and cucurbits. In WP5, tested plant extracts showed insecticidal effects against whitefly nymphs and adults comparable to commercial bio-insecticides and safer to beneficial insects. For ToBrFV, pre-infection with two mild natural and four attenuated isolates were demonstrated to protect tomato plants from ToBRFV. The long-term stability of these isolates is under evaluation. Protocols for soil treatment, including steaming at > 90°C, were optimized and proved effective in eradicating the model tobamovirus TMV. This work has increased knowledge about ToBRFV.
