Deep Sequencing Analysis Of Rnas From Citrus Plants Grown In A Citrus Sudden Death–affected Area Reveals Diverse Known And Putative Novel Viruses

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Citrus sudden death (CSD) has caused the death of approximately four million orange trees in a very important citrus region in Brazil. Although its etiology is still not completely clear, symptoms and distribution of affected plants indicate a viral disease. In a search for viruses associated with CSD, we have performed a comparative high-throughput sequencing analysis of the transcriptome and small RNAs from CSD-symptomatic and -asymptomatic plants using the Illumina platform. The data revealed mixed infections that included Citrus tristeza virus (CTV) as the most predominant virus, followed by the Citrus sudden death-associated virus (CSDaV), Citrus endogenous pararetrovirus (CitPRV) and two putative novel viruses tentatively named Citrus jingmen-like virus (CJLV), and Citrus virga-like virus (CVLV). The deep sequencing analyses were sensitive enough to differentiate two genotypes of both viruses previously associated with CSD-affected plants: CTV and CSDaV. Our data also showed a putative association of the CSD-symptomatic plants with a specific CSDaV genotype and a likely association with CitPRV as well, whereas the two putative novel viruses showed to be more associated with CSD-asymptomatic plants. This is the first high-throughput sequencing-based study of the viral sequences present in CSD-affected citrus plants, and generated valuable information for further CSD studies. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.94119CNPq, Conselho Nacional de Desenvolvimento Científico e TecnológicoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Copper Resistance Of Biofilm Cells Of The Plant Pathogen Xylella Fastidiosa

Xylella fastidiosa is a phytopathogen that causes diseases in different plant species. The development of disease symptoms is associated to the blockage of the xylem vessels caused by biofilm formation. In this study, we evaluated the sensitivity of biofilm and planktonic cells to copper, one of the most important antimicrobial agents used in agriculture. We measured the exopolysaccharides (EPS) content in biofilm and planktonic cells and used real-time reverse transcription polymerase chain reaction to evaluate the expression of the genes encoding proteins involved in cation/multidrug extrusion (acrA/B, mexE/czcA, and metI) and others associated with different copper resistance mechanisms (copB, cutA1, cutA2, and cutC) in the X. fastidiosa biofilm formed in two different media. We confirmed that biofilms are less susceptible to copper than planktonic cells. The amount of EPS seems to be directly related to the resistance and it varies according to the media where the cells are grown. The same was observed for gene expression. Nevertheless, some genes seem to have a greater importance in biofilm cells resistance to copper. Our results suggest a synergistic effect between diffusion barriers and other mechanisms associated with bacterial resistance in this phytopathogen. 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Erratum: Absence Of Classical Heat Shock Response In The Citrus Pathogen Xylella Fastidiosa (current Microbiology (2007) 54 (119b-123) Doi: 10.1007/s00284-006-0215-2)

[No abstract available]59336

MARCADORES RAPD PARA MAPEAMENTO GENÉTICO E SELEÇÃO DE HÍBRIDOS DE CITROS RAPD MARKERS TO GENETIC MAPPING AND SELECTION OF CITRUS HYBRIDS

Os marcadores moleculares apresentam várias aplicações no melhoramento de plantas, permitindo uma série de análises genéticas. Este trabalho foi realizado com o objetivo de estabelecer marcadores RAPD para serem utilizados em estudos de mapeamento genético e na seleção de híbridos entre tangerina-'Cravo' (Citrus reticulata Blanco) e laranja-'Pêra' (C. sinensis (L.) Osbeck). Extraiu-se DNA de folhas dos parentais e de seis híbridos F1. As reações de amplificação foram preparadas em 13 uL de solução, constituída por tampão 1x GIBCO BRL; soluções 1,54 mM de MgCl2 e 0,2 mM de cada dNTP; 15 ng de cada 'primer'; 1,5 unidade de 'Taq DNA Polymerase' e 15 ng de DNA genômico. As reações foram realizadas em termocicladores programados para 36 ciclos de 1 min a 92ºC, 1 min a 36ºC, 2 min a 72ºC e 10 min de extensão a 72ºC. Foram testados 'primers' decâmeros arbitrários dos 'kits' A, AB, AT, AV, B, C, D, E, G, H, M, N, P, Q, R e U da Operon, sendo selecionados 113 por apresentarem polimorfismo, com número de marcadores variando de 1 a 6 por 'primer'. Esses 'primers' amplificaram 201 (23,13%) bandas polimórficas, aplicáveis no mapeamento genético e seleção de híbridos. A freqüência de 'primers' com 1; 2; 3; 4; 5 e 6 bandas polimórficas foi de 49,5%, 33,6%, 9,7%, 4,4%, 1,8% e 1,0%, respectivamente.<br>Molecular markers have many applications in plant breeding, enabling some types of genetic analyses. The aim of this work was to establish RAPD markers to be used to genetic mapping studies and selection of hybrids between 'Cravo' tangerine (Citrus reticulata Blanco) and 'Pêra' orange (C. sinensis (L.) Osbeck). DNA of the parents and six hybrids F1 was isolated from the leaves. The amplification reactions were performed in volumes of 13 µL, composed by GIBCO BRL 1x buffer, 1,54 mM MgCl2, 0,2 mM of each dNTP, 15 ng of each primer, 1,5 unit of Taq DNA Polymerase and 15 ng of genomic DNA. These reactions were carried out in thermocyclers programmed for 36 cycles of 1 min at 92ºC, 1 min at 36ºC, 2 min at 72ºC and 10 min of extension at 72ºC. It were evaluated random decamer primers of the kits A, AB, AT, AV, B, C, D, E, G, H, M, N, P, Q, R e U from Operon. One hundred thirteen primers were selected as polymorphics, with number of markers varying from 1 to 6 per primer. These primers amplified 201 (23,13%) polymorphic fragments, with application in genetic mapping and selection of hybrids. The frequency of primers with 1, 2, 3, 4, 5 and 6 polymorphic fragments was 49,5%, 33,6%, 9,7%, 4,4%, 1,8% e 1,0%, respectively