Analysis of virome by high-throughput sequencing disclosed multiple infection in a single grapevine plant / Análise do viroma por sequenciamento de alto rendimento revelou infecção múltipla em uma planta de videira

Among phytosanitary problems of the grapevine, viruses stand out for their capacity of reducing the quality and yield of grapes. However, detecting and identifying viral infections in grapevines can be challenging. This study performed a high throughput sequencing (HTS) of the viral pathogens present in a vine showing virus-like symptoms to elucidate the etiology. HTS analysis reported in a hybrid grapevine with mild curling down of leaf edges, the presence of four viruses and viroids, which were probably implicated in the observed symptoms. The determined complete genomes showed high genetic identities with previously characterized isolates of homologous pathogens.

Dentre os problemas fitossanitários da videira, os vírus se destacam pela capacidade de reduzir a qualidade e o rendimento da uva. No entanto, detectar e identificar infecções virais em videiras pode ser um desafio. O objetivo do estudo foi realizar um sequenciamento de alto rendimento (HTS) para determinar os patógenos virais presentes em uma videira com sintomas de virose e elucidar a etiologia. Com o HTS foi detectada, em uma videira híbrida com leve enrolamento dos bordos foliares, a presença de quatro vírus e viroides, os quais provavelmente estavam implicados com os sintomas observados. Os genomas completos determinados mostraram altas identidades genéticas com isolados previamente caracterizados de patógenos homólogos.

Occurrence and richness of arbuscular mycorrizal fungi in vineyards with grapevine decline and dieback symptoms / Ocorrência e riqueza de fungos micorrízicos arbusculares em vinhedos com sintomas de declínio e morte da videira

This research identified arbuscular mycorrhizal fungi (AMF) in rhizosphere soil of grapevines with Grapevine Death and Decline symptoms (GDD) or asymptomatic healthy (H) plants, and characterized the relationship of AMF communities with soil chemical attributes. The AMF spore number ranged from 287 to 432 spores 50 cm-³ in soil with GDD plants, and from 357 to 464 spores 50 cm-³ in H plants, with no differences among vineyards or between GDD and H plants within each vineyard. We detected 42 species and 17 genera, and most taxa belonged to Acaulosporaceae or Glomeraceae. Claroideoglomus etunicatum, Funneliformis mosseae, and Archaeospora trappei were the most frequent species in all vineyards. Soil chemical attributes were not determinant for the occurrence of most fungal species; although, Entrophospora infrequens, Diversispora sp1 and Diversispora sp2 were associated with a vineyard having high soil copper. Vineyards harbor highly diverse AMF communities, which are determined by location.

Este trabalho teve como objetivo identificar fungos micorrízicos arbusculares (FMA) em solo rizosférico de videiras com sintomas de declínio e morte da videira (D) e em plantas saudáveis (S), e caracterizar a relação das comunidades de FMA com atributos químicos do solo. O número de esporos de FMA variou de 287 a 432 esporos 50 cm-³ em solo em plantas D, e de 357 a 464 esporos 50 cm-³ em plantas S, sem diferenças entre vinhedos ou entre plantas D e S dentro de cada vinhedo. Detectamos 42 espécies e 17 gêneros, sendo que a maioria dos táxons pertencia a Acaulosporaceae ou Glomeraceae. Claroideoglomus etunicatum, Funneliformis mosseae e Archaeospora trappei foram as espécies mais frequentes em todos os vinhedos. Os atributos químicos do solo não foram determinantes para a ocorrência da maioria das espécies de fungos, embora Entrophospora infrequens, Diversispora sp1 e Diversispora sp2 estivessem associados a um vinhedo com alto teor de cobre do solo. Os vinhedos abrigam comunidades FMA altamente diversificadas, que são determinadas pela localização.

Molecular characterization of GSyV-1 and GLRaV-3 and prevalence of grapevine viruses in a grape-growing area

The aims of this study were to determine the prevalence of viruses in 119 samples from 32 grapevine cultivars, collected from nine vineyards in a specific grape-growing area in southeastern Brazil, perform a partial molecular characterization of 14 isolates of Grapevine Syrah virus 1 (GSyV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3) and assess the coat protein genetic variability of these viruses. The detection of viruses was implemented by real-time RT-PCR (reverse transcription polymerase chain reaction) aiming to detect seven viruses and one viroid. With the exception of the Grapevine Cabernet Sauvignon reovirus (GCSV), the viruses and viroid that were evaluated were widespread in the sampled areas, often in high prevalence and multiple infections, ranging from 15 % up to 76 %. Eight isolates of GSyV-1 and six of GLRaV-3, partially characterized by complete coat protein gene nucleotide sequencing and a variability study showed nucleotide identities ranging from 91 % to 99 % (GSyV-1) and from 98 % to 100 % (GLRaV-3) among themselves, respectively. Comparisons between conventional and real-time RT-PCR detections were implemented for GSyV-1 and GLRaV-3 infections. Analysis of genetic variability indicated molecular differences between GSyV-1 and GLRaV-3 isolates and negative selection acting on the coat protein gene of both viruses. This is the first report of GSyV-1 in commercial vineyards in Brazil. The survey revealed widespread infections of seven important pathogens in one prominent Brazilian grape-producing region implying contaminated grapevine cuttings in the spread of disease.

Molecular characterization of GSyV-1 and GLRaV-3 and prevalence of grapevine viruses in a grape-growing area

The aims of this study were to determine the prevalence of viruses in 119 samples from 32 grapevine cultivars, collected from nine vineyards in a specific grape-growing area in southeastern Brazil, perform a partial molecular characterization of 14 isolates of Grapevine Syrah virus 1 (GSyV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3) and assess the coat protein genetic variability of these viruses. The detection of viruses was implemented by real-time RT-PCR (reverse transcription polymerase chain reaction) aiming to detect seven viruses and one viroid. With the exception of the Grapevine Cabernet Sauvignon reovirus (GCSV), the viruses and viroid that were evaluated were widespread in the sampled areas, often in high prevalence and multiple infections, ranging from 15 % up to 76 %. Eight isolates of GSyV-1 and six of GLRaV-3, partially characterized by complete coat protein gene nucleotide sequencing and a variability study showed nucleotide identities ranging from 91 % to 99 % (GSyV-1) and from 98 % to 100 % (GLRaV-3) among themselves, respectively. Comparisons between conventional and real-time RT-PCR detections were implemented for GSyV-1 and GLRaV-3 infections. Analysis of genetic variability indicated molecular differences between GSyV-1 and GLRaV-3 isolates and negative selection acting on the coat protein gene of both viruses. This is the first report of GSyV-1 in commercial vineyards in Brazil. The survey revealed widespread infections of seven important pathogens in one prominent Brazilian grape-producing region implying contaminated grapevine cuttings in the spread of disease.(AU)

Absolute quantification of viruses by TaqMan real-time RT-PCR in grapevines / Quantificação absoluta de vírus por RT-PCR em tempo real (TaqMan) em videiras

The absolute quantification determines the absolute amount of a targeted nucleic acid expressed as a copy number or concentration. The knowledge of virus concentrations in commercial crops possesses high relevance to ensure a reliable diagnosis. The objective of this study was to perform an absolute quantification of five viruses in infected grapevines (Vitis spp.). Different known amounts of the standard sample (cloned viral cDNA or in vitro transcribed viral RNA) were quantified by TaqMan RT-qPCR. Based on these data, standard curves were generated plotting Ct values (threshold cycle) against the log of the standard sample amount. Infected grapevine samples were evaluated to determine virus titers, which were highly variable. This result may contribute to improve virus diagnosis by accurately quantifying virus titre variations in grapevines.

A quantificação absoluta determina a quantidade absoluta de um ácido nucleico alvo expressa como número de cópias ou concentração. O conhecimento das concentrações virais em culturas comerciais tem grande relevância para assegurar um diagnóstico confiável. O objetivo deste trabalho foi realizar uma quantificação absoluta de cinco vírus em videiras infectadas (Vitis spp.). Diferentes quantidades conhecidas da amostra padrão (cDNA viral clonado ou RNA viral transcrito in vitro) foram quantificadas por RT-qPCR TaqMan. A partir destes dados, curvas padrão foram geradas plotando-se os valores de Ct (ciclo limiar) contra o log da quantidade da amostra padrão. Amostras de videiras infectadas foram avaliadas visando-se determinar os títulos virais que foram bastante variáveis. Este resultado contribui para melhorar o diagnóstico viral ao quantificar com precisão variações no título viral em videiras.

Absolute quantification of viruses by TaqMan real-time RT-PCR in grapevines / Quantificação absoluta de vírus por RT-PCR em tempo real (TaqMan) em videiras

The absolute quantification determines the absolute amount of a targeted nucleic acid expressed as a copy number or concentration. The knowledge of virus concentrations in commercial crops possesses high relevance to ensure a reliable diagnosis. The objective of this study was to perform an absolute quantification of five viruses in infected grapevines (Vitis spp.). Different known amounts of the standard sample (cloned viral cDNA or in vitro transcribed viral RNA) were quantified by TaqMan RT-qPCR. Based on these data, standard curves were generated plotting Ct values (threshold cycle) against the log of the standard sample amount. Infected grapevine samples were evaluated to determine virus titers, which were highly variable. This result may contribute to improve virus diagnosis by accurately quantifying virus titre variations in grapevines. (AU)

A quantificação absoluta determina a quantidade absoluta de um ácido nucleico alvo expressa como número de cópias ou concentração. O conhecimento das concentrações virais em culturas comerciais tem grande relevância para assegurar um diagnóstico confiável. O objetivo deste trabalho foi realizar uma quantificação absoluta de cinco vírus em videiras infectadas (Vitis spp.). Diferentes quantidades conhecidas da amostra padrão (cDNA viral clonado ou RNA viral transcrito in vitro) foram quantificadas por RT-qPCR TaqMan. A partir destes dados, curvas padrão foram geradas plotando-se os valores de Ct (ciclo limiar) contra o log da quantidade da amostra padrão. Amostras de videiras infectadas foram avaliadas visando-se determinar os títulos virais que foram bastante variáveis. Este resultado contribui para melhorar o diagnóstico viral ao quantificar com precisão variações no título viral em videiras. (AU)

Molecular variants of Grapevine rupestris stem pitting-associated virus infecting grapevines (Vitis spp) in Brazil / Variantes moleculares de Grapevine rupestris stem pitting-associated virus infectando videiras (Vitis spp) no Brasil

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.(AU)

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.(AU)