Characterization and epidemiology of members of the genus Torradovirus

The research described in this thesis focuses on the molecular and biological characterization of a new group of plant viruses. At the start of this research only a ‘torrado’ disease in tomato was known, which is recognised by necrosis on leaves and fruits, that eventually turn into its typical and devastating burnt-like symptoms. Using a wide range of available (virological) tools the causal agent was identified as a so-far unknown spherical virus of approximately 28 nm in diameter. Further analysis revealed that the virus contained a bi-partite RNA genome of which the entire nucleotide sequence was elucidated. The virus was named after the Spanish name of the disease, hence Tomato torrado virus (ToTV). Although this virus has been found mainly in Europe, it is emerging and meanwhile found in Central America and Australia. After the discovery of ToTV, another disease suspected to be caused by a torrado-like virus was analysed. It was shown that this disease, which was found in tomatoes grown in Mexico, was caused by a similar but distinct torradovirus named Tomato marchitez virus (ToMarV). Its molecular and biological features, as analysed and described in this thesis, supported the proposal of a new genus denoted Torradovirus, named after its first representative. This genus was recognized and classified by the International Committee on Taxonomy of Viruses (ICTV) into the family Secoviridae (order Picornavirales). Currently, the genus Torradovirus harbours, besides the recognised species ToTV and ToMarV, also the tentative species Tomato chocolate virus (ToChV), Tomato chocolate spot virus (ToChSV, analysed by another research group), and Lettuce necrotic leaf curl virus (LNLCV). Elucidation of torradovirus genome sequences revealed that their RNA1 contained one open reading frame (ORF) that contains motifs typical for proteins involved in replication, i.e. helicase, protease and RNA-dependent RNA polymerase (RdRp). RNA2, on the other hand, contained two ORFs (RNA2-ORF1 and RNA2-ORF2) that coded for a small (~20 kDa) protein of unknown function, the putative movement protein, and the three coat proteins, respectively. Considering the economic importance of torradoviruses and their emerging character, attempts were made to develop a diagnostic tool to detect the presence of these viruses. To this end generic PCR primer pairs (Torradovirus-1F/Torradovirus-1R and Torradovirus-2F/Torradovirus-2R) were developed against highly conserved regions in RNA1 and RNA2, respectively. Evaluation of these primer sets revealed that they supported detection of all currently known torradoviruses. Limited information indicated that members of the genus Torradovirus were transmitted by whiteflies. By a detailed study using varying acquisition access periods and inoculation access periods, it was demonstrated that ToTV, ToMarV and ToChV are all transmitted by three whitefly species, namely Trialeurodes vaporariorum, Trialeurodes abutilonea and Bemisia tabaci. The mode of transmission was determined as semi-persistent, i.e. like viruses that enter and remain in the foregut until being transmitted to another plant host. However, localisation of the viral RNA within the whitefly vector confirmed the presence of virus in the stylets and thereby showed that torradoviruses represent the first spherical viruses transmitted by whiteflies in a semi-persistent and stylet-borne manner.