Browsing by Author "Fonseca, Filomena"
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- Development of an asymmetric PCR-ELISA typing method for citrus tristeza virus based on the coat protein genePublication . Nolasco, Gustavo; Santos, C.; Silva, Goncalo; Fonseca, FilomenaThe coat protein gene of isolates of citrus tristeza virus (CTV) from 20 citrus-producing regions around the world was amplified by RT-PCR, TA cloned, and characterized by SSCP. Haplotypes that produced different patterns within each geographic region were sequenced and a database of 153 accessions of CTV was assembled. Phylogenetic analysis revealed the existence of seven well-defined clusters (Coefficient of differentiation 0.78). An asymmetric PCR-ELISA typing (APET) assay was developed in the frame of this clustering pattern using a set of eight hybridisation probes. The membership of any unknown haplotype is determined by comparing its pattern of reaction against the whole set of probes and not, as previously done in hybridisation assays, in an all-or-nothing basis. Interpretation of the results is objective and done through a visual basic application that compares the rates of hydrolysis of the ELISA substrate of an assayed isolate to a matrix of rates of hydrolysis obtained from standard haplotypes. This assay was validated and showed a better ability to resolve haplotypes than other assays to which it was compared experimentally. It may be automated to the same extent as any ELISA. (c) 2008 Elsevier B.V. All rights reserved.
- Digestive and metabolic consequences of on-growing greater amberjack (Seriola dumerili) juveniles at different temperatures. In-vivo and ex-vivo assessmentPublication . Navarro-Guillén, C.; Perera, E.; Pérez-Hilario, D.; Martos-Sitcha, J.A.; Molina-Roque, L.; Gregorio, Silvia; Fonseca, Filomena; Fuentes, J.; Yúfera, M.Aiming to elucidate the effects of temperature on different aspects of the fish digestive physiology that may affect the growth, we examined, firstly, variations in growth performance, feed intake, digestive enzyme activities, expression of transport related genes, metabolites in plasma and liver, and oxidative stress response in juveniles of greater amberjack (Seriola dumerili) growing during two months at 18, 22 and 26 degrees C of water temperature. Secondly, we analysed the epithelial function of the intestinal mucosa by electrophysiological ex-vivo experiments. As expected, body growth increased with increasing temperature in the tested range. Highest relative feed intake was observed at 26 degrees C, but the food conversion ratio was the same at 22 and 26 degrees C and less favourable at 18 degrees C. Digestive proteases activities were similar at 22 and 26 degrees C, while an evident effect of temperature was observed on lipids digestive capacity, being lipase activity undetectable at 18 degrees C. Electrophysiological assays revealed a relationship between temperature and intestinal mucosa plasticity. Temperature increase promoted epithelial functionality through higher tissue resistance and short-circuit current in mid-intestine at 26 degrees C, as well as better electrogenic amino acids transport. On the other hand, mRNA expression of peptide transporters tended to be higher in fish that grew at 18 degrees C, probably to reinforce the transport capacity. Plasma circulating levels of metabolites demonstrated higher energy and protein mobilization with the increasing temperature, where a hypometabolic state was denoted by lower cortisol levels at 18 degrees C together with an apparent switch from lipids to carbohydrate usage as energy source and increased oxidative stress in the liver at the lowest temperature. Altogether indicates that the tested temperatures are within the tolerance range for the species, although 22 and 26 degrees C appear as optimal temperatures for on-growing greater amberjack juveniles. Changes in nutrient digestion and absorption induced by temperature are related with both hydrolytic activity and remodelling of the intestinal mucosa. Impairing growth capacity and initial evidences of welfare compromise were observed at 18 degrees C.
- Editorial: microbiome dynamics as biomarkers of welfare status in aquatic speciesPublication . Fonseca, Filomena; Fuentes, JuanThe importance of the microbiome in aquaculture species is widely acknowledged. The fish gut microbiota is essential for host fitness, modulated by diet and environment, and affects animal physiology. The gut microbiome optimizes nutrient uptake and immunomodulation and enhances host resilience against infectious diseases. Understanding microbiome dynamics in aquaculture species is vital in promoting animal health and industry sustainability. In this Research Topic of Frontiers four original papers and one review paper were accepted. This editorial provides an integrated summary of those papers’ main discoveries and clues.
- Effects of soil drying and subsequent re-watering on the activity of nitrate reductase in roots and leaves of Helianthus annuusPublication . Azedo-Silva, João; Osório, Júlio; Fonseca, Filomena; Correia, Maria JoãoThe effects of drought on the activity of nitrate reductase (NR) were studied in Helianthus annuus L. plants subjected to soil drying and subsequent rewatering. Drought did not negatively affect the activation state of NR, but resulted in linearly correlated decreases in the activity of the unphosphorilated active form and the total activity of NR, both in roots and leaves. The concentration of nitrate in roots, xylem and leaves also decreased in waterstressed 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 waterstressed plants. Drought-induced decrease in root NR activity was 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 rewatering, 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.
- Effects of water deficit on the activity of nitrate reductase and content of sugars, nitrate and free amino acids in the leaves and roots of sunflower and white lupin plants growing under two nutrient supply regimesPublication . Correia, Maria João; Fonseca, Filomena; Azedo-Silva, João; Dias, Celeste; David, Maria Manuela; Barrote, Isabel; Osório, Maria Leonor; Osório, JúlioThe effects of a two weeks soil drying period on the activity of nitrate reductase (NR; EC 1.6.6.6) were studied on Helianthus annuus L. and non-nodulated Lupinus albus L. plants, growing under two nutrient supply regimes. NR activity was assessed in leaf and root extracts, by measuring the activity of the unphosphorylated active form (NRact), the maximal extractable activity (NRmax) and the activation state. To get insight into potential signalling compounds, nitrate, amino acids and soluble sugars concentrations were also quantified. On both species, foliar NRact and NRmax were negatively affected by soil drying and reduced supply of nutrients, the observed changes in NR activity being linearly-correlated with the depletion of nitrate. Similar results were obtained in the roots of sunflower. Conversely, in white lupin roots NRmax was found to be independent of tissue nitrate concentration. Regardless of the species and organ, the activation state of the enzyme was unaffected by the nutrient supply regime. In well-watered sunflower roots only about 50% of the existing NR was unphosphorylated, but the activation state increased significantly in response to drought. In contrast, lupin roots always exhibited NR activation state values close to 80% or even higher. At the leaf level, NR activation state was hardly changed in response to soil drying. The contribution of changes in the concentrations of soluble 5 sugars and amino acids to explain the observed variations in NR activity are discussed.
- First molecular characterization of grapevine virus B (GVB) in portuguese grapevine cultivars and improvement of the RT-PCR detection assayPublication . Fonseca, Filomena; Fernandes Duarte, Vilma Vanessa; Santos, Margarida Teixeira; Brazão, João; Eiras-Dias, EduardoThis work describes the first molecular characterization of grapevine virus B (GVB) in Portuguese grapevine cultivars. During a routine screening of 44 accessions in the National Collection of Grapevine Varieties (CAN PRT051), 17 were found infected with GVB in DAS-ELISA assays with commercial antibodies. However, only six of the corresponding isolates were successfully amplified using primer pairs described in the literature. The sequence variants (ORF4-3'UTR, 1147 nt) retrieved from these isolates segregated into two phylogenetic groups, which included sequences from complete genomes available in GenBank. The highly discrepant results obtained using serological and RT-PCR-based diagnostic tools led to the design of a primer pair for detection of GVB, which allowed the amplification of a 606-bp GVB-specific fragment from all DAS-ELISA-positive isolates and also revealed the existence of false negatives in the serological testing.
- First report of curvularia trifolii causing curvularia blight in agrostis stolonifera in South of PortugalPublication . Coelho, Luísa; Borrero, C.; Bueno-Pallero, Francisco Angel; Guerrero, Carlos; Fonseca, Filomena; Reis, Mário; Avilés, M.; Dionísio, LídiaAlgarve region (Portugal) has nearly 40 golf courses with a significant economic impact. Summer surveys on 10 golf courses detected an unknown disease on one course in 2009 and on another course in 2012 and 2013 at 29 to 30°C daily average. The second course had symptoms on about 25% of the turf of two putting greens. Diseased bentgrass (Agrostis stolonifera L.) had a green dappled pattern with irregular patches of turfgrass on yellowed leaves. Prior to decaying, affected leaves turned brown and then gray. Crown and leaf sheath infections resulted in dark brown dry rot. No lesions were observed on the roots. Leaves were surface disinfected with 5% commercial bleach (0.225% sodium hypochlorite) and cultured on potato dextrose agar (PDA). Ten fungal colonies grew from the leaf tissue, and brown mycelia, conidiophores, and conidia were observed under a microscope. Conidia were ventricose pyriform, mostly abruptly curved, 20 to 36 µm (30 µm, SD = 4) × 7 to 12 µm (10.5 µm, SD = 1.3) (n = 50), predominantly three-septate, with a prominent hilum and enlarged and darkened central cells. Colonies grown on PDA were black-brown with a black reverse side. Conidia differed in size, 15.4 to 24.6 µm (19.99 µm, SD = 3.00) × 6 to 11 μm (8.68 µm, SD = 1.54) (n = 50) and morphology (cylindrical or slightly curved). These characteristics were consistent with Curvularia trifolii (Kauffm.) Boedijn. (Ellis 1971; Falloon 1976; Khadka 2016). Species identification of the representative isolate A2 1.12 was confirmed by analysis of nucleotide sequences of the ITS1-5.8S-ITS2 region using primers ITS1 and ITS4 (White et al. 1990) and GPDH gene region with primer set gpd (Koike et al. 2013). BLAST searches of GenBank showed a high similarity of the isolate ITS sequence (MG029439) to the reference sequence JN712458 of C. trifolii (99% identity) and GPDH sequence (MK570108) with LT715803.1 (97.88% identity). The maximum likelihood phylogenetic tree showed that our isolate clustered with C. trifolii. The pathogenicity assay of this isolate was conducted in greenhouse on A. stolonifera ‘Penncross’. The isolate was grown on PDA (25°C, 10 days). Five pots (100 ml) were filled with a sand and peat mix (9:1 v/v) with 0.06 g of seeds per pot, covered with a fine sand layer. Turfgrass was cut once a week beginning 2 weeks after seeding and was fertigated with 0.5 g/liter of Peter’s foliar feed (27 + 15 + 12; N + P2O5 + K2O; and micronutrients; Scotts, Heerlen, The Netherlands). To obtain a conidial suspension for inoculation, cultured plates were scraped with a sterilized spreader and water. The suspension was filtered through a sterile gauze. Conidia were counted under a microscope (400×) with a hemocytometer. The suspension was adjusted to 8 × 103 conidia/ml, and 10 ml was sprayed per pot. Pots maintained humidity for 2 days under microtunnels. The first disease symptoms appeared 3 days after inoculation. Bentgrass from the five pots developed Curvularia blight and rotted crown symptoms. Control plants (five pots treated with water) did not display symptoms. This trial was repeated once. On PDA, C. trifolii was reisolated from leaf lesions and morphologically identified, confirming Koch’s postulates. Ellis (1971) referred to the presence of C. trifolii in Portugal, but no region, symptom description, or grass species was detailed. Sivanesan (1987) reported C. trifolii in Portugal only on Lolium multiflorum. Therefore, this is the first report of C. trifolii in Algarve, affecting A. stolonifera. This disease can increase maintenance costs in greens in this area.
- From invasion to fish fodder: inclusion of the brown algae rugulopteryx okamurae in aquafeeds for european sea bass dicentrarchus labrax (L., 1758)Publication . Fonseca, Filomena; Fuentes, Juan; Vizcaíno, Antonio Jesús; Alarcón, Francisco Javier; Mancera, Juan Miguel; Martínez-Rodríguez, Gonzalo; Martos-Sitcha, Juan AntonioIn keeping with the premises of Blue Circular Economy in the European Union, the present study explored the possibility of using the invasive brown alga Rugulopteryx okamurae in aquafeeds for European sea bass (Dicen-trarchus labrax). Assuming the raw algae biomass could negatively impact animal performance, four experi-mental formulations were prepared, by including macroalgae material at 5%, using crude (CR), enzymatically hydrolysed and fermented (EF), enzymatically hydrolysed (E), or fermented (F) R. okamurae biomass, which we tested against a control feed (CT). To evaluate the effects of the experimental diets, besides animal growth performance and biometric parameters, we devised a toolbox focused on the intestine and intestinal function: i) ex-vivo epithelial resistance and permeability in Ussing chambers; ii) microbiota composition through NGS; iii) expression profiles of selected markers for epithelial integrity, transport, metabolism, and immune response, by qPCR. Our results show differentiated allometric growth among diets, coupled with intestinal epithelium al-terations in permeability, integrity, and amino acid transport. Additionally, evidence of microbiota dysbiosis and contrasting immune responses between experimental diets, i.e. pro-inflammatory vs. anti-inflammatory, are also described. In conclusion, we believe that R. okamurae could be a suitable resource for aquafeeds for the European sea bass, although its use requires a pre-treatment before inclusion. Otherwise, while the fish still have a positive growth performance, the gastrointestinal tract pays a toll on the integrity, transport, and inflammatory processes.
- Genetic variants of Grapevine leafroll-associated virus 2 infecting Portuguese grapevine cultivarsPublication . Fonseca, Filomena; Esteves, Filipa; Teixeira Santos, Margarida; Brazão, João; Eiras-Dias, José EduardoGenetic variability of 19 isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) from Portuguese grapevine cultivars was characterized by sequencing the entire capsid protein (CP) gene of the virus. Global phylogenetic analysis of the CP gene, which included nucleotide sequences obtained in this study and complete homologous sequences from GenBank, showed segregation of GLRaV-2 variants from Portuguese isolates into three major phylogroups (PN, 93/955 and H4). The novelty of these phylogenetic results is the evidence of well-supported subdivision within H4 as well as within PN, with subgroup PN3 composed exclusively of variants from a Portuguese isolate. These findings and the genetic analysis of global phylogroups indicate demographic expansion, mainly within PN and 93/955. Because the existence of a mixture of variants from different phylogroups was detected in some of the isolates, a typification assay based on reverse transcription reaction followed by polymerase chain reaction and restriction fragment length polymorphism analysis, was developed to complement molecular detection assay of the virus. This protocol discriminates variants from the phylogroups identified in this study, and is appropriate for routine testing for GLRaV-2.
- Genomic variability of Citrus tristeza virus (CTV) isolates introduced into MoroccoPublication . Lbida, B.; Fonseca, Filomena; Santos, C.; Zemzami, M.; Bennani, A.; Nolasco, GustavoGenomic 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.
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