VIRVITIS - Avaliação Molecular e Estratégias Aplicáveis ao Controlo do Urticado e Enrolamento Foliar da Videira

Domain: Agricultural Sciences

Grapevine fanleaf virus (GFLV) and Grapevine leafroll-associated viruses (GLRaVs) -1 and 3 are the main etiological agents, involved respectively in Grapevine infectious disease (GID) and Grapevine leafroll disease (GLD), the major viral diseases of grapevines worldwid. Current control strategies are preventive, designed to keep viruses separated from their vectors, but diseases are spreading and threatening grapevine industry.

BACKGROUND: The development of resistance/protection in rootstocks and/or cultivars against the implicated viruses continues to be a major challenge due to the limited efficacy of current measures and the increasing demand for sustainable and environmentally safe agricultural practices. Contribution from fundamental research on the molecular characterization of the implicated viruses has been sought and applied. The molecular evidence on GFLV has allowed the production of transgenic rootstocks expressing the viral capsid protein (CP) gene or specific viral antibodies. Testing of these scientific accomplishments in open field trials however, has faced delays due to public rejection (20) preventing the assessment of the stability of the obtained resistance. A less controversial application of molecular work on GFLV has been the identification and successful use of mild strains for cross-protection in lab and field tests. Regarding GLD, and because more etiological agents are involved, with different genomic structures, molecular research has still to identify and characterize genomic variants to be able to make a contribution similar to GID. There are currently 9 recognized GLRaVs, but complete genome sequences have been submitted only for GLRaV-1, -2 and -3. Recently we obtained new CP sequences for GLRaV-1 and GLRaV-3 showing the existence of distinct CP variants for each virus. On the basis of this, we produced novel antibodies and successfully tested this new tool with an in situ immuno-assay (ISIA). Clearly more molecular work is needed to fully characterize virus species and variants involved in GLD.

Given the current state of the art and public opinion, resistances are unlikely to be based upon genetically modified plants and other sources have to be found/developed. Interactions between virus genomic variants or strains need to be unraveled to identify mild strains suitable to use in cross-protection. To understand the mechanisms enabling cross-protection and make this an effective control strategy, further molecular characterization of the etiological agents involved is necessary as well as to explore virus-host interactions and related changes in host virus-mediated genetic expression.

OBJECTIVES: In agreement with the points discussed above and based on the work and tools we have been developing, the objectives of our proposal are threefold:

1- To assess viral incidence and detect virus variants present in grapevines grown in Portugal, subjected to field conditions with and without phytosanitary control, in order to determine stability and dynamics of variability in both situations.

2- To molecularly characterize the genomes of molecular variants found for GFLV, GLRaV-1 and GLRaV-3 in order to conduct an extensive in silico analysis that will reveal aspects of virus phylogeny and evolutionary processes.

3- To study and dissect GFLV, GLRaV-1 and -3 –host interactions adequate to produce a functional analysis of virus variants and their potential for use in cross-protection, through identification of candidate genes for virus-mediated changes in host expression.

APPROACH:The national ampelographic collection (CAN, established in 1988) will be a source of material infected with GFLV, GLRaV-1 and -3, following previous screening work (PTDC/AGR-AAM/65094/2006), as well as material contributed by grapevine extension services (MADRP). The viral molecular characterization will develop so that complete sequences for the variant types involved are obtained. Results from this work shall provide a sound molecular basis for phylogenetic inference and establish the molecular evidence needed to choose the source material for the ensuing biological characterization. Each variant group will be characterized, in terms of virus- host interactions at the genetic expression level, after transference to an indicator. Progress of infection shall be followed through fluorescent ISIA. The Suppression Subtractive Hybridization (SSH) method will be used to probe for genes with virus-mediated differential expression. All information combined will allow the characterization of virulence and provide a functional analysis of virus variants and virus-host interactions.

IMPACT: This work will provide molecular tools for probing resistance of grapevine, as well as a molecular basis for identifying virus variants suitable for cross-protection.