Browsing by Author "Nolasco, Gustavo"
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- A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogensPublication . Nolasco, Gustavo; Deblas, C.; Torres, V.; Ponz, F.A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA. is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been succesfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.
- Asymmetric PCR ELISA: increased sensitivity and reduced costs for the detection of plant virusesPublication . Nolasco, Gustavo; Sequeira, Z.; Soares, C.; Mansinho, A.; Bailey, A. M.; Niblett, C. l.PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan(TM), a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the costs of PCR ELISA without reducing its sensitivity. The usefulness of these modifications was demonstrated for the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus. We expect that with only minor modifications asymmetric PCR ELISA could be used effectively for the detection of most nucleic acid molecules of interest.
- Bacterial expressed coat protein: development of a single antiserum for routine detection of Citrus tristeza virusPublication . Sequeira, Z.; Nolasco, GustavoCitrus tristeza virus (CTV) causes one of the most important citrus diseases world-wide and has recently been detected in Portugal. Early diagnosis based on immunoenzymatic techniques requires significant amounts of reagents. This research describes the isolation of the coat protein gene of CTV, its expression in Escherichia coli as a fusion protein containing an N-terminal (His)6 region and its use to raise rabbit polyclonal antibodies. These antibodies could be used at dilutions higher than 1/80,000 at the detection stage in indirect ELISA tests and were also efficient for trapping the virus in ELISA and Immunocapture RT-PCR. These characteristics allowed the production of diagnostic kits based solely on this source of antibodies. A detection spectrum and sensitivity similar to that of a commercial polyclonal antibody kit was achieved.
- Biological characterization of Citrus tristeza virus monophyletic isolates with respect to p25 genePublication . Hancevic, K.; Cerni, S.; Nolasco, Gustavo; Radic, T.; Djelouah, K.; Skoric, D.Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus and displays a high level of genetic and phenotypic diversity. In this study the biological characterization of monophyletic CTV-isolates based on p25 gene (Gp 1, Gp 2, Gp 3a, Gp 4, Gp 5, Gp M) was analyzed for the first time on the set of standard indicator plants and unde r the same environmental conditions, in order to compare the phenotypic characteristics of p25 genomic variants. The results showed that tested CTV-isolates varied in their ability to induced symptoms as well as in severity of symptoms e. g. pathogenicity. (C) 2012 Elsevier Ltd. All rights reserved.
- Can bicarbonate enhance the performance of carob seedlings grown in nutrient solutions with different Fe concentrations?Publication . Gama, Florinda; Correia, Pedro José; Saavedra, T.; Dandlen, Susana; de Varennes, Amarilis; Nolasco, Gustavo; Pestana, MaribelaThe aim of this work was to assess the effect of bicarbonate (Bic) ion on the nutritional status and performance of carob-tree seedlings, a species that normally grows in calcareous soil without exhibiting iron chlorosis symptoms. Seedlings were previously grown in nutrient solution with a small concentration of Fe (0.5-1 mu M) to induce a moderate chlorosis. Afterwards, two experiments were established: in experiment 1, plants were grown for 21 days in the following treatments: Fe deficiency (Fe0), 0.5 mu M Fe, 5 mu M Fe, and 5 mu M Fe plus calcium carbonate (CaCO3). After assessing these results, a second experiment was conducted for 91 days, with the following treatments: Fe0, 1 mu M Fe, 40 mu M Fe and 40 mu M Fe plus CaCO3 and sodium bicarbonate (NaHCO3). Chlorophyll of young leaves, biomass and mineral composition of leaves, stems and roots were assessed in both experiments. The ferric chelate reductase root activity (FC-R) and the genetic expression of calmodulin-regulated Ca2+-ATPase pump (ACA gene) were evaluated in experiment 2. Fe-deficient plants exhibited reduced growth and enhanced macronutrients in leaves. Root micronutrient homeostasis changed as an adaptive mechanism in carob. The addition of bicarbonate did not aggravate Fe chlorosis, as leaf chlorophyll increased significantly. Root FC-R activity and ACA gene expression was not enhanced under Fe deficiency induced by bicarbonate (Fe40 + BicNa) which suggest a positive effect of bicarbonate in the metabolism of this crop. Nevertheless, small Fe concentrations (Fe1) induced a higher ACA gene expression thus indicating some stress response signalling.
- Cellular location of Prune dwarf virus in almond sections by in situ reverse transcription-polymerase chain reactionPublication . Silva, C.; Tereso, S.; Nolasco, Gustavo; Oliveira, M. M.In situ reverse transcription-polymerase chain reaction (RT-PCR) was used in young leaves (from trees and in vitro shoots) and flower buds of almond (Prunus dulcis), a stone fruit, for cellular location of Prune dwarf virus (PDV, a member of the genus Ilarvirus). Sections obtained from samples fixed in formaldehyde and embedded in paraffin were refixed in formaldehyde to increase tissue preservation in the RT-PCR steps. The coat protein gene of PDV was used as the target to produce a cDNA copy that was amplified by PCR and visualized using a direct detection method with digoxigenin-labeled nucleotides. Protein digestion, PCR, and detection strategies were optimized for increased tissue preservation and signal intensity. PDV was found in infected samples within the vascular tissue of young leaves and flower buds as well as in the mesophyll in developing floral organs and in the generative and vegetative cells of pollen grains. PDV signals were observed in a ring surrounding the nucleus and spread in the cytoplasm. The results obtained are discussed in terms of the technique optimization and PDV distribution in tissues and transmission through pollen. The optimized protocol of in situ RT-PCR is a powerful technique to reveal low-abundant RNA species. Therefore, it is appropriate to study cell and subcellular distribution of RNA viruses in woody species.
- Citrus tristeza virus p23 may suppress systemic silencing but is not related to the kind of viral syndromePublication . Costa, A.; Marques, N T.; Nolasco, GustavoCitrus tristeza virus (CTV) the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins namely p20, p23 and p25 protein, previously reported for the monophyletic isolate T36. In this study the p23 suppressing activity from isolates of each of seven phylogenetic groups recognized for CTV was characterized in Nicotiana benthamiana 16C plants expressing GFP. Data revealed that the p23 protein from each phylogenetic group was able to transiently suppress the local but not the short-range silencing. p23 from Group 5 was the most efficient local suppressor followed by Groups 1, 3a, 3b and 4; the Groups 2 and M were less efficient. Different development of systemic silencing was observed among p23 proteins. This was most conspicuous for Gp 5, which completely blocked the GFP systemic silencing, suggesting that a quantitative relationship might exist between local and systemic silencing. A 3D model of the p23 protein was constructed showing differences within the Zn-finger region, which, however, do not totally explain the differences found. It was not possible to trace a relationship between the syndromes tribute to the various phylogenetic groups and p23 activity. (C) 2014 Elsevier Ltd. All rights reserved.
- Citrus variegation virus: Molecular variability of a portion of the RNA 3 containing the coat protein gene and design of primers for RT-PCR detectionPublication . Bennani, B.; Mendes, Celso; Zemzami, M.; Azeddoug, H.; Nolasco, GustavoThe coat protein gene and part of the intergenic region of the RNA 3 of several isolates of Citrus variegation virus (CVV) producing either infectious variegation or crinkly leaf symptoms were amplified by RT-PCR, cloned and sequenced. Some isolates were composed of a mixture of sequence variants. The coat protein gene appeared to be highly conserved (lowest similarity among all CVV sequences 93%), especially at the N-terminal, indicating at the molecular level that both types of symptoms are indeed produced by the same virus species. No relationship could be found with the geographic origin. Sequences obtained from isolates producing infectious variegation clustered in a different branch of a dendrogram than those originating from crinkly leaf symptoms. Both clusters could further be distinguished by two parsimonious sites in the coat protein gene. In the short stretch of the intergenic RNA 3 region analysed, a stable hairpin exists in addition to the previously reported hairpin that constitutes the core promoter for the RNA 4 transcription. This second hairpin could also be recognised in the other subgroup 2 Ilarviruses. Surprisingly, at the nucleotide and amino acid levels and in the secondary features of the intergenic region, CVV appeared closer to the other subgroup 2 Ilarviruses than to Citrus leaf rugose virus to which it is serologically related and has been considered to be evolutionary related. Using primers designed for the conserved regions, the virus was detected with a prevalence of 25% and 13% in Portuguese and Moroccan citrus collections. A group of RT-PCR positives was further confirmed by ELISA and biological indexing.
- Citrus wilsonii: Biological response to infection with different citrus tristeza virus genotypesPublication . Hancevic, K.; Cerni, S.; Nolasco, Gustavo; Radic, T.; Rosin, J.; Gatin, Z.; Skoric, D.Citrus wilsonii Tanaka is cold-resistant, rarely grown chance hybrid between Citrus ichangensis and Citrus grandis with a potential to be used as a rootstock in colder climates. In order to evaluate its reaction to Citrus tristeza virus (CTV), C. wilsonii seedlings were graft-inoculated with previously characterized CTV isolates monophyletic with respect to the p25 gene and clustering to phylogenetic groups Gp 1, Gp 2, Gp 3a, Gp 4, Gp 5 and Gp M. The evaluation of C. wilsonii symptoms and comparison with the reaction of standard citrus indicators infected with the same CTV isolates revealed that C. wilsonii infected with isolates Gp 2 and Gp 4 developed stem pitting (SP). This is in accordance with the SP-inducing potential of these CTV isolates in the standard sweet orange SP indicator. The obvious and numerous pits shown by C. wilsonii stems suggests that it reacts strongly to severe SP CTV isolates.
- Co infection of OMMV and OLV-1 enhances symptoms and increases both viruses accumulation and viral derived siRNAs in plantsPublication . Varanda, C. M.; Materatski, P.; Campos, M. D.; Clara, M. I.; Marques, N T.; Nolasco, Gustavo; Felix, M. R.Previous extensive field surveys in olive orchards have revealed high levels of Olive mild mosaic virus (OMMV) and Olive latent virus 1 (OLV-1), frequently appearing in mixed infections. These viruses belong to genus Alphanecrovirus and their RNA dependent RNA polymerase (RdRp),as well as their p6 and p8 amino acid sequences share over 87% identity.