Filomena Fonseca

Effects of soil drying and subsequent re-watering on the activity of nitrate reductase in roots and leaves of Helianthus annuus

The effects of drought on the activity of nitrate reductase (NR) were studied in Helianthus annuus L. plants subjected to soil drying and subsequent re-watering. Drought did not negatively affect the activation state of NR, but resulted in linearly-correlated decreases in the activity of the unphosphorylated active form and the total activity of NR, in both roots and leaves. The concentration of nitrate in roots, xylem and leaves also decreased in water-stressed plants, whereas the concentration of total amino acids was only transiently depressed at the leaf level. In contrast, soluble sugars accumulated both in roots and leaves of water-stressed plants. Drought-induced decreases in root NR activity were correlated with the observed changes in root nitrate concentration. A higher percentage of the decrease in foliar NR activity could be explained by the decline in nitrate flux to the leaves than by leaf nitrate content. Following re-watering, the extent of recovery of NR activity was higher in roots than in leaves. The delay in the recovery of foliar NR activity did not result from the persistence of reduced flux of nitrate through the xylem. Several hypotheses to explain the after-effect of soil drying on foliar NR activity are discussed.

Genomic variability of prune dwarf virus as affected by agricultural practice

Summary.Twelve new sequences of the coat protein gene of Prune dwarf virus (PDV) variants, obtained from almond trees, are presented. Comparison with previously reported sequences of the same region, obtained from other hosts (plum, cherry and peach) revealed not only the existence of a wider range of variants of PDV than formerly predicted, but also the frequent presence of a mixture of variants in each sample. In spite of the heterogeneity found in almond, the amino acid composition of the domain at the N terminus of the coat protein maintained the potential to form an amphipathic helix, and hence the capacity to serve the previously suggested function of binding the viral RNA during particle formation.Except for synonymous substitutions, measures of nucleotide diversity calculated for the two groups, respectively 13 sequences from almond and 14 sequences from other hosts, were found to be significantly different, with the almond group showing a much higher variability. Analysis of the dendrogram constructed based in all 27 PDV CP sequences did not reveal host specificity, in agreement with previous findings. However, a clear divergence between almond and other hosts sequences could be found. It is discussed that the observed differences between almond and other hosts variants may derive from differences in agricultural practices.

Molecular data mining to improve antibody-based detection of Grapevine leafroll-associated virus 1 (GLRaV-1).

Testing for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.

Genomic variability of Citrus tristeza virus (CTV) isolates introduced into Morocco

Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single stranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomic variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each isolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates characterised in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyer lemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coat protein gene sequence is useful to trace the origin of the isolates.

Occurrence of grapevine leafroll-associated virus 5 in Portugal: genetic variability and population structure in field-grown grapevines

A comparison of 15 field isolates of grapevine leafroll-associated virus 5 (GLRaV-5) was conducted, based on the analysis of nucleotide sequences of two viral ORFs: the coat protein (CP) and the heat shock protein 90 homolog (HSP90h). After amplification and cloning, the population of viral sequences was analyzed for each isolate, revealing the within-isolate presence of sequence variants for both genes, with one or more major CP variants. Phylogenetic analysis showed the gene sequence variants detected to be exclusive for each isolate. These data, together with estimates of genetic diversity and positive selection, did not reveal evidence of vector-mediated transfer of GLRaV-5. Conversely, a strong effect of host vegetative propagation on divergence dynamics of GLRaV-5 variants was suggested by the isolates from this work The phylogeny of the CP gene further revealed clustering of GLRaV-5 isolates into eight lineages, four of which were detected in our study, revealing a higher diversity than previously described. The information gathered also contributes to firmly establishing GLRaV-5 as a cohesive taxonomic group within the ampeloviruses.

Typing of egyptian Citrus tristeza virus (CTV) isolates based on the capsid protein gene

The capsid protein gene of three Egyptian CTV isolates from two locations was amplified by immunocapture RT-PCR and analysed by single stranded conformation polymorphism and sequencing. The CTV isolates studied did not differ significantly in sequence composition and each isolate consisted of very similar haplotypes. Comparison with reference sequences from isolates elsewhere in the world showed that these haplotypes clustered very close to the severe strain T3 from Florida causing quick decline and stem pitting. Analysis of the deduced amino acid sequence showed the epitope characteristic of reactivity with the MCA13 antibody. Sequence comparison with the sequence of an Egyptian isolate (Qaha) available in the Genbank showed a distance of about 8%, suggesting that it had a different origin.