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  • Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3
    Publication . Gouveia, P.; Dandlen, S. A.; Costa, A.; Marques, N T.; Nolasco, Gustavo
    GLRaV-3, a member of the Closteroviridae family and type member of the genus Ampelovirus, is involved in the grapevine leafroll disease. Until now no RNA silencing suppressor has been found among viruses of this genus, contrary to what happens with a large number of other viral genera. In the sister genus Closterovirus, RNA silencing suppressors are present in the 3' end of the genome and have molecular weights close to 20 KDa. To test for RNA suppressing activity screening of p21, p19.6 and p19.7 proteins, coded for in an analogous genomic location of the GLRaV-3 was undertaken. Only p19.7 revealed suppressor activity demonstrated in diverse silencing inducing systems. This suppressor is able to overcome strong silencing inducers and shares several properties with the BYV p21-like family of suppressors of the closteroviruses. This is the first report of an RNA silencing suppressor in the genus Ampelovirus.
  • Gene transcripts responsive to drought stress identified in Citrus macrophylla bark tissue transcriptome have a modified response in plants infected by Citrus tristeza virus
    Publication . da Silva, Melina; Pinto, Patricia IS; Guerra, Rui Manuel Farinha das Neves; Duarte, Amilcar; Power, Deborah; Marques, N T.
    Citrus macrophylla Wester (CM) has valuable agronomic characteristics such as the ability to grow in saline soils, although with low tolerance to prolonged drought stress (DS). To understand the mechanisms that characterize CM response to water scarcity, this study compared transcriptome profile changes in CM stem tissue when exposed to DS and identified a total of 2745 differentially expressed transcripts (DETs, fold change > 2), of which 631 were up-regulated and 2114 were down-regulated. DETs up-regulated by DS were enriched in pathways such as the redox and osmotic system or soluble carbohydrates and in transcripts for low molecular weight proteins such as late embryogenesis abundant protein (LEA). Down-regulated transcripts were mainly assigned to photosynthesis, transport, phenylpropanoids, calcium dependent kinases, brassinosteroids and other hormones including salicylic acid and abscisic acid. To assess the interplay between DS and Citrus tristeza virus (CTV) infection, twelve genes were profiled by quantitative Real-Time PCR (qPCR) analysis in control and CTV-infected CM plants, with or without DS. The twelve analyzed transcripts were significantly correlated (r = 0.82, p < 0.001) with the RNA-Seq results and gave insight into the responses of CM to drought and/or to infection with CTV. Transcriptome results unveiled highly responsive genes to DS in stem tissue, which may be candidates for genetic selection of high drought tolerant plants of CM.
  • Poda de Citrinos na Região Mediterrânica. Manual Técnico
    Publication . Barrote, Isabel; Neto, Luís; Guerrero, Carlos; Marques, Natália; Duarte, Amilcar
    A poda é uma prática comum em citrinos, mas que é por vezes questionada, por se tratar de uma operação dispendiosa e por as árvores não podadas terem produções razoáveis. No entanto, a poda tem provado ser de particular importância na citricultura mediterrânica, orientada para a produção de frutos para consumo em fresco. Nesta citricultura a poda permite aumentar a qualidade do fruto, a qual é altamente valorizada, pelo que a poda se torna necessária e é compensada pelo preço de venda dos frutos. A poda permite formar e controlar a copa da árvore para obter uma melhor produtividade e qualidade dos frutos e, simultaneamente, melhorar o estado sanitário da árvore e facilitar a execução de operações, como a colheita e os tratamentos fitossanitários. Neste manual, resumimos e explicamos as técnicas de poda utilizadas na citricultura mediterrânica e referimos os principais objetivos de cada tipo de poda, tendo em consideração a morfologia e a fisiologia dos citrinos. Este livro é o resultado de vários anos de trabalho em ensaios de poda realizados no Algarve e foi escrito no âmbito do Grupo Operacional “PodaCitrus - Otimização da poda em citrinos”, financiado por fundos da União Europeia, através do programa PDR2020. É, assim, o produto de um vasto trabalho de equipa, que, além dos autores, incluiu muitos agrónomos, responsáveis técnicos pelos pomares onde foram realizados os ensaios. Foi também importante a participação de agrónomos exteriores ao projeto, com quem discutimos os resultados obtidos e as técnicas de poda de citrinos praticadas nos campos. Os autores agradecem, em primeiro lugar, às empresas Frusoal, Citriaroeira, Produção Citrícola Lda, e João Santana Unipessoal, parceiras do projeto, mas também às empresas Quinta da Barragoa e Valenciagro (grupo Martinavarro) as respetivas colaborações. Pessoalmente, agradecemos a Silvino Oliveira, Angélica Mendonça, Valter Reis, Manuel Reis, Marta Afonso, Rui Antão, Tiago Guerreiro, Vera Sustelo, Luís Mendonça e Alfonso Barrau, toda a sua colaboração. Agradecemos também a Gonçalo Azinheira pela elaboração das ilustrações deste livro e a Miguel Santos pela edição de duas fotografias. Agradecemos ainda a Hugo Marques e a Vera Sustelo pela disponibilização de algumas fotografias.
  • Differentiation of Citrus tristeza virus (CTV) isolates by cleavase fragment length polymorphism (CFLP) analysis of the major coat protein gene
    Publication . Marques, N T.; Bailey, A. M.; Niblett, C. L.; Nolasco, Gustavo
    A panel of Citrus tristeza virus (CTV, genus Closterovirus, family Closteroviridae) isolates of different origins and with different biological properties were compared for polymorphisms in the major coat protein (CP) gene by cleavase fragment length polymorphism (CFLP) and single stranded conformation polymorphism (SSCP) analysis. The similarity between the CFLP patterns, which consisted of 15 to 20 bands, was estimated by the Pearson coefficient. The clustering patterns from the CFLP data were very similar to those from sequence data in an experiment with 16 cloned standards of the CP gene. By SSCP analysis on the other hand, most of the clones were not clustered in the same way. To assess the ability of CFLP to analyse biological samples, which may consist of a mixture of genomic variants, the CP gene of 12 CTV isolates was obtained directly from infected plants by immunocapture/RT-PCR and analysed. With few exceptions, the isolates were correctly clustered according to the sequences of the variants composing the isolates. In artificial mixed infections of mild and severe isolates the patterns obtained were more closely related to the severe isolate. Thus the CFLP method was an accurate method for the identification, typing and clustering of CTV isolates. The usefulness of this technique as an alternative to SSCP analysis is suggested and discussed.
  • Callose synthase and xyloglucan endotransglucosylase gene expression over time in Citrus × clementina and Citrus × sinensis infected with citrus tristeza virus
    Publication . da Silva, Melina; Germano, Sandra; Duarte, Amilcar; Pinto, Patrícia; Marques, Natália
    Citrus tristeza virus (CTV) is a virus that already caused great losses in citrus producing regions. The cell wall of plant cells plays an important role in the defence response to viruses. Following several studies indicating that cell wall enzyme transcripts of callose synthase 7 (calS7) and xyloglucan endotransglucosylase 9 (xth9) are modified during a viral infection, transcript expression of calS7 isoform x5 (calS7x5) and xth9 was evaluated over time in Citrus x sinensis 'Valencia Late' (VL) and Citrus x clementina 'Fina' (CL), infected with the severe CTV isolate T318A, by quantitative (q) PCR. qPCR analysis of healthy and CTV infected citrus was performed at 15 days, 10 months and at 31 months post-inoculation (dpi/mpi), respectively. The CTV titer, evaluated at the three time-points by qPCR, increased over time in bark tissues, with VL plants exhibiting a titer about 5 times higher than CL 31 mpi. CTV infection did not cause significant changes in calS7x5 gene expression over time in both citrus cultivars. However, CTV infection was associated with significant up-regulation of xth9 in VL compared to controls 31 mpi. This study highlights that CTV infection can affect the expression of specific cell wall-associated genes over time and that this influence was distinct for VL and CL. This study provides further insight into the CTV-citrus host interaction, with the long-term response of VL to a severe CTV isolate involving a high expression of the xth9 gene.
  • Pitaya as a new alternative crop for Iberian Peninsula: Biology and edaphoclimatic requirements
    Publication . Trindade, Ana Rita; Paiva, Paulo; Lacerda, Vander; Marques, Natália; Neto, Luís; Duarte, Amilcar
    Pitaya is one of the fruit species whose demand has increased in recent years due to the numerous health benefits and lucrative price of the fruit and its by-products. In Europe, the Iberian Peninsula and other Mediterranean countries are the ones with favorable climatic conditions for its cultivation. This document describes much of the history of pitaya in the Iberian Peninsula and the difficulties related to its cultivation. A bibliographical survey was carried out on the culture of pitaya in the world, focusing on the edaphoclimatic requirements, and on the possibility of this becoming a consolidated crop in the Iberian Peninsula. The relatively low water requirement of pitaya makes this crop sustainable among crops that require irrigation. In addition, we provide a perspective for use and research of this emerging crop. There has been an exponential growth of scientific publi-cations on pitaya in the last decade; however, much more needs to be researched to know how to increase productivity as well as the sensory quality of fruits in different regions. This sustainable crop is a good option to diversify fruit production in the Iberian Peninsula.
  • Identification of asymptomatic plants infected with Citrus tristeza virus from a time series of leaf spectral characteristics
    Publication . Afonso, Andreia; Guerra, Rui Manuel Farinha das Neves; Cavaco, A. M.; Pinto, Patricia IS; Andrade, André; Duarte, Amílcar; Power, Deborah; Marques, N T.
    Citrus tristeza virus (CTV) affects citrus crops with differing severity, depending on the viral strain, the citrus cultivar and the scion/rootstock combinations. In this study we address the problem of identifying asymptomatic infected plants using reflectance spectra of the leaves in the visible/near infrared region. Sixteen young citrus plants (8 Citrus x clementina hort. ex Tanaka ‘Fina’ and 8 Citrus sinensis (L.) Osbeck ‘Valencia Late’) were split into control and T318A isolate infected groups. Measurements of reflectance in the 400-1100 nm range, in two leaves per plant, were performed monthly over 6 months and the presence of the virus was confirmed by IC/RT-PCR and real-time PCR. The spectra acquired in a single day of measurements was inconsistent for inoculated and control plants. However, by monitoring the same leaves over 6 months it was possible to identify infected plants on the basis of the spectra time evolution. In order to achieve this a simple unfolding implementation of 3-way PCA was applied such that group separation in the scores plot was spontaneous and not forced by any a priori assumption. The model was tested through leave-one-out cross validation with a good rate of correct classification for the left out sample. A real situation was simulated by applying the NPCA algorithm to healthy plants only and checking if the infected ones would be projected on the model scores plot as outliers. Again, a good rate of classification was obtained. Finally, we discuss the spectral features that may be associated with the clustering obtained through NPCA and their physiological significance. Reflectance measurements between infected and healthy samples of two citrus cultivars and their correlation with real-time PCR results for the presence of CTV suggest reflectance spectra of the leaves in the visible/near infrared region is a promising tool for plant stress monitoring linked to the presence of CTV infection prior to symptom expression.
  • Citrus aphids in Algarve Region (Portugal): Species, hosts, and biological control
    Publication . Paiva, Paulo Eduardo Branco; Neto, Luis; Marques, Natália; Duarte, Beatriz; Duarte, Amilcar
    Aphids affect citrus by causing leaf deformations and reducing fruit production. Additionally, aphids are a great concern due to their ability to transmit Citrus tristeza virus (CTV), the cause of tristeza, one of the main citrus diseases. In the last four years, citrus orchards in the south of Portugal (Algarve region) were sampled for aphid species identification and counting. Aphis spiraecola was the most abundant species, representing more than 80% of all identified aphids, and the damage (leaf deformation) it causes was directly proportional to its density. A. gossypii was the second most common species, followed by A. aurantii and Macrosiphum euphorbiae. The number of aphids in nymph stages was predominant over the adult stages (both wingless and winged) in all species. A. citricidus, the most efficient CTV vector, was not detected. The largest populations of A. spiraecola were observed in lemon and orange trees during spring (>100 individuals per shoot), with great damage observed in orange, lemon, and mandarin trees. A. gossypii was observed mainly in mandarin and tangor trees. There was a low activity of natural biological control agents, with the parasitism of A. spiraecola by Lysiphlebus spp. and Binodoxys spp. ranging from 0.3 to 1.5%. The numerical ratio ranged from 150 to 440 aphids per predator, and among these, syrphids were the most abundant, followed by lacewings and coccinellids (Scymnus).
  • Citrus tristeza virus p23 may suppress systemic silencing but is not related to the kind of viral syndrome
    Publication . Costa, A.; Marques, N T.; Nolasco, Gustavo
    Citrus 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.
  • Comparing p20's RNA silencing suppressing activity among five phylogenetic groups of Citrus Tristeza virus
    Publication . Marques, N T.; Costa, A. A.; Lopes, D.; Silva, Goncalo; Nolasco, Gustavo
    The p20 protein encoded by the Citrus Tristeza Virus (CTV) was previously identified as a RNA silencing suppressor. In this study, we analyzed the p20's suppressing activity from five phylogenetic groups of CTV, using the co-infiltration assay of Green fluorescence protein (GFP) gene and the suppressor gene in 16C line Nicotiana benthamiana plants. Green fluorescence, GFP mRNA relative levels and GFP specific siRNAS were compared showing in most cases, only slight differences. Contrary to previous studies, the p20 suppressor was not able to impede neither short range nor systemic spreading of RNA silencing. The suppressor from the phylogenetic group 4 revealed a much reduced activity when compared with the others. At present we still don't know whether this property is a characteristic of this group or an atypical feature due to a unique point mutation. The differences in the symptom type and intensity originated by isolates belonging to the phylogenetic groups assayed could not be related to differences to the p20 suppressor's activity.