Molecular characterization and development of new diagnostic methods for the genus Fabavirus. BTH evaluation as a control method.

	The genus Fabavirus of the family Comoviridae includes three species: Broad bean wilt virus 1 (BBWV-1), BBWV-2 and Lamium mild mosaic virus (LMMV), although recently a new species, Gentian Mosaic Virus (GeMV), has been proposed. These viruses infect a large number of economically important horticultural and ornamental species and are transmitted in a non-persistent mode by twenty different aphid species. The genome is composed of two single stranded positive-sense RNA molecules that are encapsidated separately by two coat proteins to form icosahedral virions. BBWV-1 and BBWV-2 are distributed worldwide. In Spain, BBWV-1 was found in pepper crops in Aragón, Cataluña, Castilla-León, C. Valenciana, Murcia, País Vasco and La Rioja. 
	Fabaviruses constitute a poorly characterized viral group. At the beginning of this work, the complete nucleotide sequences of only six BBWV-2 isolates from East Asia and partial sequences of a few BBWV-1 and BBWV-2 isolates were available. Therefore, obtaining the complete sequence of at least one BBWV-1 isolate and partial sequences of other isolates from different geographical origins was considered a necessary step to develop improved detection methods taking account of the genetic variability of the virus.
	In this thesis, the complete RNA sequence of the BBWV-1 Spanish isolate 1S1 was determined. The genome is composed of two single-stranded and positive-sense RNA molecules of 5814 and 3431 nt, with the genomic organization characteristic of members of the family Comoviridae. Comparison with the complete sequence of the American isolate 1U2 of BBWV-1 (published during the progress of this  work) showed high divergence along the genome, with nucleotide and amino acid identities around 80% and 93%, respectively.
	Sequence alignment of the two BBWV-1 isolates, the six BBWV-2 isolates and one GeMV isolate were used to design four different conserved primer pairs that were used to reverse transcribe (RT) and PCR amplify partial sequences of BBWV-1, BBWV-2 and GeMV isolates collected worldwide. Analysis of these sequences and others available at GenBank revealed high genetic diversity of BBWV-1 and BBWV-2 in comparison with other plant viruses. Also, our analysis showed that recombination, selective pressure along the genome and long distance migration have played a role in the genetic structure and evolution of these viruses. 
	New detection methods based on molecular hybridization (dot-blot and tissue-print formats) or RT-PCR were developed and. their sensitivity for virus detection in different hosts and plant tissues compared with ELISA. Thus, a pair of conserved primers were designed that enabled diagnosis of fabavirus infections in a single RT-PCR reaction and specific identification of GeMV, BBWV-1 and BBWV-2 even in mixed infections, based on the size of the amplification products. Also, we designed a method for universal detection of BBWV-1 or BBWV-2 by molecular hybridization with probes corresponding to a conserved genomic region, and discrimination of isolate groups or strains, using probes corresponding to a more variable genomic region. 
	Finally, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) was evaluated as a potential control agent. Application of BTH in Arabidopsis thaliana plants previously inoculated with BBWV-1 induced a delay on virus accumulation (detected by RT-PCR and molecular hybridization) the and symptoms onset . BTH is environmentally respectful and it has a wide spectrum action, two characteristics that could make it appropriate for fabavirus control, considering the high variability of these viruses.