Molecular variants of Grapevine rupestris stem pitting-associated virus infecting grapevines (Vitis spp.) in Brazil.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most common viruses of grapevine. It is involved in the graft-transmissible disease rupestris stem pitting of the rugose wood complex. The objective of the research was to perform the molecular characterization of the coat protein (CP) gene of sixteen Brazilian GRSPaV isolates aiming to determine the occurrence of molecular variants (strains) of this virus. Nine grapevine samples were evaluated, from which dsRNA was extracted. Nucleotide sequences were generated by Next generation sequencing (NGS). Fifteen complete sequences of the GRSPaV CP gene were obtained and phylogenetically analyzed. Multiple alignments of the sequences showed identities of nucleotides ranging from 82% to 99%, suggesting high variability among the CPs of Brazilian isolates. The study revealed that genetic variability of GRSPaV comprising three molecular variants is also present in Brazilian grapevine genotypes.Key words: GRSPaV, Foveavirus, strains, lineages, genetic variability. O GRSPaV é um dos vírus mais comuns da videira. Está associado à doença transmissível por enxertia denominada caneluras de rupestris que compõe o complexo do lenho rugoso. O objetivo do trabalho foi realizar a caracterização molecular do gene da proteína capsidial (CP) de 16 isolados brasileiros de GRSPaV visando determinar a ocorrência de variantes moleculares desse vírus. Nove amostras de videira foram avaliadas das quais foi extraído dsRNA. As sequências de nucleotídeos foram geradas pelo sequenciamento de nova geração (NGS). Quinze sequências completas do gene CP de GRSPaV foram obtidas e filogeneticamente analisadas. Os alinhamentos múltiplos entre as sequências mostraram identidades de nucleotídeos variando de 82% a 99%, sugerindo alta variabilidade entre as CPs de isolados brasileiros. O estudo revelou que a variabilidade genética de GRSPaV compreendendo três variantes moleculares também está presente nos genótipos de videira no Brasil. Palavras-chave: GRSPaV, Foveavirus, estirpes, linhagens, variabilidade genética

Leaf hyperspectral reflectance as a potential tool to detect diseases associated with vineyard decline.

Grape production in the Serra Gaúcha region, south of Brazil, is severily constrained by several diseases such as the decline and death syndrome caused grapevine trunk (fungal) diseases (GTDs) and the grapevine leafroll-associated virus (GLRaV). As pathogens induce changes in leaf tissue that modify the reflectance, the spectral signature of asymptomatic and symptomatic grapevine leaves infected by GTDs and GLRaV was analyzed to check whether spectral responses could be useful for disease identification. This work aims at (a) defining the spectral signature of grapevine leaves asymptomatic and symptomatic to GTDs and GLRaV; b) analyzing whether the spectral response of asymptomatic leaves can be distinguished from symptomatic; and (c) defining the most useful wavelengths for discriminating spectral responses. For such, reflectance of leaves in either condition collected in a ?Merlot? vineyard during three growing seasons was measured using a spectroradiometer. Principal components and partial least square discriminant analyses confirmed the spectral separation and classes discrimination. The average spectra, difference spectra, and first-order derivative (FOD) spectra indicated differences between asymptomatic and symptomatic leaves in the green peak (520?550 nm), chlorophyll-associated wavelengths (650?670 nm), red edge (700?720 nm), beginning of nearinfrared (800?900 nm), and shortwave infrared. Hyperspectral data was linked to biochemical and physiological changes described for GTD and GLRaV. Variable importance in the projection (VIP) analysis showed that some wavelengths allowed to differentiate the tested pathosystems and could serve as a basis for further validation and disease classification studies. Keywords Grapevine leafroll-associated virus . Grapevine trunk diseases . Vitis vinifera L. . Principal components analysis . Variable importance in the projectio

Detection and partial molecular characterization of isolates of Grapevine leafroll-associated virus 4 and Grapevine rupestris vein feathering virus.

Resumo apresentado de número 371-2

Role of the 5' untranslated region of the Alfalfa mosaic virus RNA 3 in cell-to-cell and long distance transport.

Após o início da infecção de uma única célula, vírus de planta necessita para invadir as células adjacentes, uma processo de transporte de célula-a-célula denominados, como um passo anterior para invadir as partes distais do hospedeiro através do sistema vascular ou do transporte sistémico. A capacidade para atingir as partes não inoculadas de um planta implica que o vírus deve infectar específico células localizadas no tecido vascular. Na maioria dos casos, partículas do vírus são necessários para este trans- vascular port. No presente estudo, tratamos o caracterização de determinantes virais críticos para o transporte de longa distância usando o Alfalfa vírus do mosaico (AMV) sistema de modelo, o que exige partícula do vírus para o transporte sistêmicaEdição dos Resumos do 12º Congreso Nacional de Virología, Burgos, Espanha, jun. 2013

Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus.

O Rupestris stem pitting-associated virus (RSPaV) é o agente causal das caneluras do lenho da videira. Este trabalho teve como objetivo produzir antissoro policlonal a partir da proteína capsidial (CP) recombinante do RSPaV e avaliar a sua especificidade e sensibilidade. O gene da CP do RSPaV, com 780pb, foi previamente caracterizado. Esse gene foi subclonado no sítio de restrição EcoRI, no vetor de expressão pRSET-B e o plasmídeo recombinante foi utilizado para induzir a expressão da CP em Escherichia coli. A CP, ligada a uma cauda de seis histidinas, foi purificada por meio de cromatografia de afinidade em coluna de Ni-NTA a partir do extrato de proteínas totais extraídas de E. coli. A identidade da proteína purificada foi confirmada em SDS-PAGE e Western blot, utilizando-se anticorpos comerciais contra a cauda de seis histidinas. A CP recombinante expressada in vitro apresentou massa molecular de cerca de 31kDa. A proteína purificada foi quantificada e 2,55mg foram utilizados para a imunização de um coelho. O antissoro policlonal obtido reagiu com diferentes isolados deste vírus, extraídos de videiras em ELISA indireto.Nota técnica

Advances in propagation of grapevine in the world.

Grapevine production by classical grafting methods and in commercial scale emerged over 130 years. This system remained handmade until the mid-1950s, when emerged the first international certification programs aimed at obtaining mother lants with high viral sanity. The necessity to increase the scale of production on industrial model and plant material production based on minimum morphological standards appeared at the end of the 1960s. Along the 1970s, research unlocked knowledge on emi-automated grafting, process hygiene, use of plant growth regulators and understanding of physiological events of rootstock-scion compatibility, callus formation and rooting. So, until the mid-2000s, certification schemes and propagation processes advanced little in technical standard. However, grapevine growing areas were expanded and demands for plant material increased, and new diseases emerged from contaminated nurseries. These new diseases (new viral complexes, phytoplasmas, bacteria and grapevine trunk diseases) were discovered by high-sensitivity diagnostic methods. Today, there is a new discussion on the nursery segment worldwide. The propagation techniques have been reviewed from the perspective of reducing the incidence of new diseases and minimum physiological damage of nursery plants during the production stages. Therefore, technological innovations regarding equipment, practices and production inputs have been incorporated in new certification schemes. However, despite these dvantages, these schemes have become more complex and multidisciplinary than previous ones, bringing difficulties in adaptation of nurserymen. Index-terms: Vitis grafting, nursery, certification, grapevine cuttings. Propagação de videiras a partir dos métodos clássicos de enxertia e em escala comercial teve origem há mais de 130 anos. Este sistema permaneceu artesanal até meados da década de 1950, quando se iniciaram os primeiros programas internacionais de certificação com foco na obtenção de plantas básicas com elevada sanidade para vírus. No fim da década de 1960, surgiu a necessidade de aumentar a escala para produção em um modelo industrial em que a muda apresentasse um padrão morfológico mínimo. Ao longo da década de 1970, aprofundaram-se as pesquisas relacionadas à automatização da prática de enxertia, à higienização do processo, ao uso de reguladores de crescimento e ao entendimento dos eventos fisiológicos da compatibilidade entre enxerto e porta-enxerto, formação de calos e enraizamentos. Assim, até meados dos anos 2000, os esquemas de certificação e o processo de propagação pouco evoluíram em termos técnicos. Porém, a medida que a área vitícola foi expandindo e a demanda por mudas aumentando, verificou-se que novas doenças se alastravam em escala global a partir de viveiros contaminados. Estas novas doenças (complexos virais, fitoplasmas, bactérias e fungos causadores de podridões vasculares) foram sendo descobertas à medida que os métodos de diagnose avançaram em sensibilidade de detecção. Hoje, surge nova discussão no segmento viveirista mundial fundamentada no fato de que o processo de propagação está sendo revisto sob o foco da redução de incidência das novas pragas e mínimo dano à muda ao longo das etapas da produção. Surgem, assim, inovações tecnológicas, tanto em equipamentos quanto em práticas e insumos, sendo incorporadas aos novos modelos de certificação. Mas, se por um lado, estes esquemas tornam-se cada vez mais multidisciplinares, por outro, a complexidade gerada pode trazer dificuldades para a adesão pelos viveiristas. Termos para indexação: Vitis enxertia, viveiro, certificação, mudas de videira

Grapevine viruses survey in Pernambuco, Paraiba and Bahia states, Brazil.

Resumo apresentado de número 371-1

Discovery and molecular characterization of a novel enamovirus, Grapevine enamovirus-1.

In this study, we describe a novel putative Enamovirus member, Grapevine enamovirus-1 (GEV-1), discovered by high-throughput sequencing (HTS). A limited survey using HTS of 17 grapevines (Vitis spp.) from the south, southeast, and northeast regions of Brazil led to the detection of GEV-1 exclusively on southern plants, infecting four grapevine cultivars (Cabernet Sauvignon, Semillon, CG 90450, and Cabernet franc) with a remark-able identity of around 99% at the nucleotide level. This novel virus was only detected in multiple-virus infected plants exhibiting viral-like symptoms. GEV-1 was also detected on a cv. Malvasia Longa by RT-PCR. We performed graft-transmissibility assays on GEV-1. The organization, products, and cis-acting regulatory elements of GEV-1 genome are also discussed here. The near complete genome sequence of GEV-1 was obtained during the course of this study, lacking only part of the 3 0 untranslated terminal region. This is the first report of a virus in the family Luteoviridae infecting grapevines. Based on its genomic properties and phylogenetic analyses, GEV-1 should be classified as a new member of the genus Enamovirus . Keywords High-throughput sequencing Virus discovery Luteoviridae Grapevine enamovirus-1 GEV-

Plant height and hydraulic vulnerability to drought and cold

Understanding how plants survive drought and cold is increasingly important as plants worldwide experience dieback with drought in moist places and grow taller with warming in cold ones. Crucial in plant climate adaptation are the diameters of water-transporting conduits. Sampling 537 species across climate zones dominated by angiosperms, we find that plant size is unambiguously the main driver of conduit diameter variation. And because taller plants have wider conduits, and wider conduits within species are more vulnerable to conduction-blocking embolisms, taller conspecifics should be more vulnerable than shorter ones, a prediction we confirm with a plantation experiment. As a result, maximum plant size should be short under drought and cold, which cause embolism, or increase if these pressures relax. That conduit diameter and embolism vulnerability are inseparably related to plant size helps explain why factors that interact with conduit diameter, such as drought or warming, are altering plant heights worldwide

Simultaneous detection of the 13 viruses and 5 viroids affecting grapevine by molecular hybridization using a unique probe or 'polyprobe'.

Grapevine is the most economically important fruit crop in the world, which is affected by various diseases of viral and/or viroidal etiology, which may affect the production of grapes with losses of up to 15% (Martelli, 1993)