Abstract Pepino mosaic virus (PepMV) is an important tomato-infecting virus which is widely spread throughout the tomato crops worldwide, where produces important economical losses. PepMV has an efficient mechanical transmission which leds to its quick spread and makes difficult the control of the disease. The high variability in symptom expression and large genetic diversity of this virus complicates the diagnosis. Nowadays five genotypes of PepMV are characterized although the genome region which is responsible of symptom expression remains unknown. Full-length infectious clones of the virus and the constructions of mutant clones marked with fluorescent proteins have been demonstrated as an efficient tool for studying the molecular biology of RNA viruses. Likewise, the interaction of PepMV with the fungal vector Olpidium brassicae (Wor.) Dang sensu lato was associated with the disease referred to as “tomato collapse”, however the possible transmission of the virus by this fungal vector was never studied. Therefore, a diagnosis method to allow the identification and simultaneous detection of the five described genotypes of PepMV: European (EU), Peruvian (PE), Chilean 1/US1 (CH1/US1), Chilean 2 (CH2) and US2 was developed. The method consisted in firstly perform a one step multiplex RT-PCR with a cocktail of six specific primers which amplified a fragment of the RNA-dependent RNA-polymerase, plus an internal control, differentiating three groups of genotypes: EU/PE, CH1/US1 y CH2/US2. To identify the concrete genotype within the groups a restriction analysis with the endonuclease SacI was carried out to the obtained PCR product. The detection limit and sensitivity of this method was higher when compared to DAS-ELISA and molecular hybridization, common techniques used for virus detection. On the other hand, a full-length clone of an isolate of the CH2 genotype of PepMV was constructed. The derived in vitro RNA transcripts of this clone were inoculated to Nicotiana occidentalis H-H Wheeler resulting in virus infection and causing indistinguishable symptoms from those of the wild-type isolate. Later, a mutant clone of this isolate was constructed with the green fluorescent protein (GFP) gene inserted in the intergenic region between ORF 4 and 5 of the PepMV genome. To verify the possible transmission of PepMV by the fungal vector O. brassicae sl (specifically O. virulentus (Sahtiyanci) Karting), two experiments were perfomed in a growth chamber. PepMV was transmitted to healthy tomato plants by irrigation with the drainage water obtained from PepMV-infected plants and whose roots contained the fungal vector O. virulentus isolated originally from tomato. Furthermore, in 2007 a new virus named Tomato torrado virus was identified as the causal agent of the “torrado” disease. Therefore, some aspects of the epidemiology, diagnostic methods, variability and possible synergic effects with other viruses which commonly affect Spanish tomato crops should be clarified. Firstly, to determine the occurrence and distribution of the torrado disease in the main Spanish tomato growing areas from 2001 to 2008, a total of 584 samples from symptomatic and asymptomatic plants were collected from 92 greenhouses of different areas were infected with ToTV. Samples were classified by symptoms and analysed to different viruses. More than 75% of the collected samples were infected with ToTV. The majority of the positive samples showed typical symptoms of the disease; however plants showing different symptoms of necrosis or even asymptomatic plants were infected with the virus. Co-infection of ToTV with Pepino mosaic virus (PepMV) occurred in a large number of samples (60.5%), and several samples were infected with other tomato-infecting viruses. Moreover, the tissue-printing hybridization is a reliable technique which could facilitate the routine diagnosis and large-scale analysis of ToTV. Additionally, weed species found in the surrounding area of the tomato crops from Murcia, Tenerife and Gran Canaria were sampled and analysed against ToTV. Twenty-two samples from different botanic genera were positive to the virus. Later, samples showing single-infection with ToTV and mixed-infected with other tomato-infecting viruses found in plants affected by torrado disease were selected. Optical and electron microscopy studies were conducted to know the cytopathology induced in tomato leaves single-infected by ToTV and the effect of the co-infection of ToTV with other viruses which commonly infect tomato crops. Ultra-thin sections of ToTV-infected tomato leaves did not present a strong cellular alteration. However, crystalline arrays of isometric virus-like particles (VLPs) of 20-30 nm in the inclusion bodies were observed in phloem parenchyma cells of the infected tissues. Tissues double-infected by ToTV and Tomato chlorosis virus (ToCV) or Pepino mosaic virus (PepMV) presented more severe cellular alterations. The most deleterious consequences for tomato cells were found in triple infections of ToTV, PepMV and Tomato spotted wit virus (TSWV), where characteristic cell wall overgrowth was distinguishable, together with a large amount of necrotic cells. Finally, the population structure and genetic variation of Tomato torrado virus (ToTV) were estimated from 19 Spanish isolates collected from 2001 to 2009 in different tomato production areas by analyses of the partial nucleotide sequences of five regions of the virus genome: the protease-cofactor and the RNA-dependent RNA-polymerase, the movement protein and two subunits of the coat protein (Vp35 and Vp23). In the analysis, three Hungarian isolates of the virus were also included. All the ToTV isolates clustered together in the phylogenetic analysis of the nucleotide sequences of the different regions. A high similarity was observed among all the isolates studied. However, some genetic diversity was observed in case of subunits of the CP studied among the Gran Canary isolates and the rest of analysed isolates of ToTV that grouped together. Studying the genetic distances between pairs of sequences, the ratio between nonsynonymous and synonymous substitutions was low, indicating a strong negative selective pressure in the studied regions. Nine negatively selected sites distributed in different areas of the genome and just one site in the protease-cofactor under positive selection for all the genome regions studied were found. ?? ?? ?? ?? Abstract________________________________________________________________ viii vii Abstract