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- Gene transcripts responsive to drought stress identified in Citrus macrophylla bark tissue transcriptome have a modified response in plants infected by Citrus tristeza virusPublication . 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.
- O Huanglongbing (HLB) no Brasil: o que se conseguiu desde 2004Publication . Paiva, Paulo; Duarte, AmílcarA citricultura brasileira tem sido acometida por várias doenças, muitas delas, limitantes. Na década de 1930 a ocorrência do vírus da tristeza dos citrinos (CTV) causou a morte de cerca de dez milhões de árvores enxertadas em laranjeira azeda. Duas décadas depois, a introdução da bactéria Xanthomonas axonopodis pv. Citri, causadora do cancro cítrico motivou a criação de um programa nacional de erradicação desta doença, mantido no estado de São Paulo até recentemente. A erradicação nunca foi alcançada mas manteve a doença com baixa incidência. Anomalias relacionadas com porta-enxertos como o declínio dos citrinos (citrus blight) na década de 1970 e a morte súbita dos citrinos, no final da década de 1990, ambas sem causa conhecida, foram controladas com a substituição de porta enxertos suscetíveis por tolerantes. Atualmente, duas doenças têm alto custo de controlo: uma virose de laranjeiras doces causada pelo vírus da leprose dos citros (CiLV) e transmitida pelo ácaro Brevipalpus phoenicis e a pinta preta dos citrinos (citrus black spot), cujo agente causal é o fungo Guignardia citricarpa. Para além dessas doenças, o surgimento da clorose variegada dos citrinos (CVC) na década de 1980 mudou a citricultura brasileira. A CVC, causada pela bactéria de xilema Xylella fastidiosa e transmitida por 12 espécies de cigarrinhas, obrigou a que a produção de plantas (mudas) fosse feita em ambientes protegidos de insetos vetores. Em 2000 toda a formação de plantas estava em estufas. Além disso, adotou-se o sistema de cultio em substrato, em bancadas, para evitar a contaminação com Phytophthora spp. e fitonematóides. Como as plantas mães (matrizes) já estavam certificadas e protegidas, assegurou-se a sanidade do material de propagação.
- Callose synthase and xyloglucan endotransglucosylase gene expression over time in Citrus × clementina and Citrus × sinensis infected with citrus tristeza virusPublication . da Silva, Melina; Germano, Sandra; Duarte, Amilcar; Pinto, Patrícia; Marques, NatáliaCitrus 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.
- Identification of asymptomatic plants infected with Citrus tristeza virus from a time series of leaf spectral characteristicsPublication . 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.
- An efficient procedure to extract RNA from a single Aphis gossypii and Aphis spiraecola for detecting Citrus tristeza virus by nested RT-PCRPublication . Dandlen, Susana Anahi; Russo, Guilherme; Horta, Soraia; Duarte, Amilcar; Paiva, Paulo; Tomás Marques, NatáliaA simple protocol to extract RNA from a single wingless aphid, Aphis gossypii and Aphis spiraecola, using liquid nitrogen and Tri-Reagent is described. A nested reverse-transcription polymerase chain reaction with degenerate primers enabled Citrus tristeza virus (CTV) detection in these aphid species collected from an infected tangor ‘Ortanique’ (Citrus × sinensis (L.) Osbeck × Citrus reticulata Blanco) in the field. The procedure enabled a CTV detection rate of 75% in the A. gossypii and A. spiraecola evaluated.