Detection and molecular characterization of Grapevine yellow speckle viroid 1 isolates infecting grapevines in Brazil

Abstract

Presently, Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the only viroids reported to infect grapevines (Vitis spp.) in Brazil, among the seven viroid species already reported infecting this host in other countries. All grapevine viroid diseases are graft-transmissible and can induce losses especially when associated with viruses. The aim of this work was to confirm infection by Grapevine yellow speckle viroid 1 (GYSVd-1) in grapevine samples exhibiting yellow speckle symptoms in the leaves and in asymptomatic samples sequenced by next generation sequencing (NGS). The occurrence of this viroid in Brazil was further investigated in a second study. Total RNAs and dsRNAs were extracted from five symptomatic plants and 16 asymptomatic samples, respectively. Specific primers were used for RT-PCR and amplified DNA fragments were cloned and sequenced by the Sanger method. Eleven complete nucleotide sequences of GYSVd-1 isolates (366-367 nt) were obtained from NGS and from RT-PCR amplicons. Comparisons showed high identities (95.9-100 %) among ten isolates and an identity of 87.2-90.4 % with a divergent isolate (RM-BR). Phylogenetic analyses placed GYSVd-1 isolates in four clusters (types 1, 2, 3 and 4). All GYSVd-1 infections were confirmed by conventional RT-PCR and RT-qPCR using specific oligonucleotides and a labeled probe. This is the first report and molecular characterization of GYSVd-1 infecting grapevines in Brazil, and our survey indicates that this viroid could be widespread in the major grape producing regions of Brazil.

First report of Grapevine Pinot gris virus infecting grapevine in Brazil

“…Appropriated controls were included in all performed analyses. RT-qPCR reactions were carried out using the kit TaqMan® Fast Virus 1-Step Master Mix (Life Technologies) in a thermocycler StepOnePlus Real-time PCR System (Applied Biosystems) as previously described (Fajardo et al 2016). Reaction data were analyzed using the StepOne Software v.2.3 (Applied Biosystems) by determining the Ct (threshold cycle).…”

“…Two sets of primer pairs were used to amplify the complete CP of GPGV, DetF (Saldarelli et al 2015) and GPgV588R (Bianchi et al 2015), and the partial RNA dependent RNA polymerase (RdRp) domain of the GPGV replicase gene, GPGVRepF and GPGVRepR (Saldarelli et al 2015). Amplification cycling, cloning and purification of recombinant plasmids from Escherichia coli were performed as described by Fajardo et al (2016). Automatic nucleotide sequencing was performed with two clones per amplicon and isolate.…”

First report of Hop stunt viroid infecting Vitis gigas, V. flexuosa and Ampelopsis heterophylla

“…HSVd, Citrus exocortis viroid (CEVd), and Grapevine yellow speckle viroid 1 (GYSVd-1) have been detected in diseased grapevines in Brazil (Eiras et al 2006;Fajardo et al 2016). Information about variability of HSVd isolates from Brazilian grapevines is limited (Eiras et al 2006).…”

“…400 ng). RT-PCR reactions and amplification cycling, amplified DNA analysis, elution of DNA fragments, cloning, purification of recombinant plasmids from Escherichia coli and nucleotide comparisons were performed as described by Fajardo et al (2016). The Sanger nucleotide sequencing was performed using two clones per isolate.…”

“…The viral and viroidal communities in the samples included, at least, 16 other pathogens: Grapevine enamovirus-1, Grapevine Cabernet Sauvignon reovirus, Grapevine vein clearing virus, Grapevine Red Globe virus, Grapevine Syrah virus 1, Grapevine leafroll-associated virus 2, 3, 4 and 4 strain 5, Grapevine rupestris stem pitting-associated virus, Grapevine virus A, B and D, Grapevine fleck virus, Grapevine rupestris vein feathering virus, and GYSVd-1 (Fajardo et al 2016. It was not possible to associate specific symptoms or disorders with HSVd infections because all positive viroid samples also included various combinations of the above mentioned viruses.…”