ABSTRACT
       
       
       
       
       Plant viruses cause important economical losses in many agricultural crops worldwide. Virus infection is a very complex process consisting of replication and expression of the virus genome within the host cell, cell-to-cell and systemic movement of the virus, successful countering of host plant defenses and transmission to other plants. This involves numerous specific interactions of viruses with their plant hosts, vectors and often with other viruses co-infecting the same plant. Disease control is mainly based on prophylactic measures to avoid or hinder virus spread, such as certification and quarantine programs, removal of infected plants when virus incidence is low, agronomical practices, etc; as well as the use of resistant cultivars obtained by plant breeding or genetic engineering. This requires rapid, specific and reliable methods of virus detection and identification and for some tasks a quantification method to assess virus accumulation, e.g. evaluation of plant resistance. However, disease control is often inefficient or is overcome in a short time due to the great ability of viruses for rapid evolution and adaptation to new environments. This entails that viral diseases are very dynamic processes with frequent cases of emergence (very rapid increase of virus incidence) with devastating consequences.
       The knowledge of factors involved in virus evolution is crucial for understanding the processes involved in virus molecular biology and epidemiology (including emergence), as well as developing more efficient and durable strategies for disease control. The goal of this thesis is to gain insight on how some factors involved in the virus life cycle can affect the fitness and determines the evolution of two viruses: Broad bean wilt virus 1 (BBWV-1, genus Fabavirus, family Secoviridae) and Tobacco mosaic virus (TMV, genus Tobamovirus, family Virgaviridae).
	First, the necessary tools for these studies were developed. Flow-through hybridization of tissue-prints allowed BBWV-1 detection in only 30 min without processing the samples. Real time quantitative RT-PCR (RT-qPCR) was developed for BBWV-1 and the other important member of the genus Fabavirus, Broad bean wilt virus 2 (BBWV-2). They enabled absolute quantification of divergent isolates of BBWV-1 and BBWV-2 with a wide dynamic range and high sensitivity (1000-10000 viral RNA molecules). Also, full-length cDNA BBWV-1 genomic RNAs were constructed from which infectious transcripts were generated, which showed the same infectivity (percentage of infected plants), viral accumulation and symptomatology as did the BBWV-1 isolate from which the clones were constructed.
	These tools were used to study several factors affecting BBWV-1 fitness. Analysis of time-course accumulation of BBWV-1 showed a temporal pattern consisting of an exponential increase of viral titer reaching a maximum, followed by a decrease until a minimum peak and a another increase until reaching a plateau with small fluctuations. This pattern was observed in Vicia faba and Chenopodium quinoa plants, but the accumulation in the later was delayed. Dilutions of the inoculum doses (1/10 and 1/100) and inoculation of plants with different development stages had little effect on the virus accumulation. Application of Acibenzolar-S-methyl (BTH), an activator of systemic acquired resistance (SAR), produced a decrease of BBWV-1 infectivity but did not affect viral accumulation in the infected plants. This effect was stronger when the doses and application times were increased. Coinoculation of BBWV-1 and Tomato spotted wilt virus (TSWV) in V. faba plants produced a hypersensitive response (HR) triggered by TSWV which prevented or retarded systemic infection of TSWV and produced a drastic decrease of infectivity of BBWV-1 (menor o igual al 15%) with respect to plants singly inoculated with BBWV-1 (mayor o igual al 80%) and plants preinoculated with TSWV (60%). In spite of the infectivity reduction, BBWV-1 accumulation was unaffected by the coinoculation with TSWV in the few plants where BBWV-1 was detected. Transmission assays of two BBWV-1 isolates with nine aphid species using different virus source plants revealed that the viral titer in the leaf where the aphid acquired the virus was correlated to the transmission rate. No differences in transmissibility between both BBWV-1 isolates were observed when the viral accumulation in the source plant was considered. However, some aphid species transmitted BBWV-1 more efficiently than others.
       Finally, fitness of TMV and another member of the genus Tobamovirus, Oilseed rape mosaic virus (ORMV) were evaluated in four A. thaliana mutants: mutants tor1 and tor2, with the cellular microtubule dynamics altered, and mutant npr1, and cpr5 with low and high plant defense level (SAR) respectively. ORMV accumulation seemed not to be affected by these mutations whereas a drastic decrease of TMV accumulation occurred in cpr5 plants. To determine if TMV could adapt to these mutants an evolutionary experiment was performed consisting of serial passages of TMV in two A. thaliana ecotypes corresponding to six genotypes: ecotype Ler (wild type, mutant tor1 and mutant tor2) and ecotype Col-0 (wild type, mutant npr1 and mutant cpr5). Nucleotide analysis of the genomes of the resulting virus lineages showed 12 amino acid substitutions and 8 synonymous nucleotide substitutions in 12 lineages with respect to the ancestors of these lineages. Time course evaluation of infectivity and viral accumulation of these lineages in the A. thaliana wild type and mutants suggested that the A. thaliana mutants tor1 and specially tor2 could have induced genetic changes in TMV affecting infectivity and/or viral accumulation. No effect or little effect was observed for the npr1 and cpr5 TMV evolved lineages.