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

Abstract

ABSTRACT: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 realtime 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.

High-throughput sequencing applied for the identification of viruses infecting grapevines in Brazil and genetic variability analysis

“…The virus GVB (ORF1 -replicase) had the higher π value (0.22880 ± 0.02406). The lowest π value was detected in GLRaV-3 coat protein gene (0.03846 ± 0.00780), consistent with previous work (Moura et al 2017). In general, the π values detected in this work are in accordance with π values found by other authors in studies involving viruses with DNA and RNA genomes (Garcia-Arenal et al 2001;Lima et al 2013;Zanardo et al 2014a;Moura et al 2017).…”

“…GSyV-1 was first identified in USA (Al Rwahnih et al 2009) and symptoms include swollen graft unions, cracking and pitting of the wood, stem necrosis, leaf reddening and scorching, vine decline and death. Its presence has been reported in many countries (Glasa et al 2015) and was recently reported infecting vineyards in Brazil (Moura et al 2017). GVCV was found associated with leaf vein-clearing, shortening of the internodes, crinkled, misshapen and smaller leaves and reduced grape berry size, with irregular shape and abnormal texture (Guo et al 2014;Zhang et al 2011) and GRGV was previously reported in many viticultural countries (Beuve et al 2015).…”

“…Watterson's estimator for the population-scaled mutation rate (Θ-W) for most of the analyzed viruses was in the order of 10 −2 (except for the RdRP of GLRaV-2 and GLRaV-4 strain 5; CP of GLRaV-4; partial polyprotein of GRGV; ORF1 of GRSPaV and GVB; and MP of GVA and ORF 1, for which it was in the order of 10 −1 ) (Supplementary Table S3). The Θ-W values of 10 −1 are lower when compared with other coding regions and other viral species described in the literature (Lima et al 2013;Moura et al 2017;Rocha et al 2013;Zanardo et al 2014a). It is noteworthy that datasets with few sequences may mask the results of variability descriptors, over-or underestimating the genetic variability.…”

“…In general, the following viruses were found in mixed infections: GCSV, family Reoviridae; GFkV and GRGV ( Ty m o v i r i d a e , genus M a c u l a v i r u s ) ; G L R a V-2 (Closteroviridae, Closterovirus); GLRaV-3, GLRaV-4 and GLRaV-4 strain 5 (Closteroviridae, Ampelovirus); GRSPaV (Betaflexiviridae, Foveavirus); Grapevine rupestris vein feathering virus (GRVFV) and GSyV-1 (Tymoviridae, Marafivirus); GVA, GVB and Grapevine virus D (GVD) (Betaflexiviridae, Vitivirus); and GVCV (Caulimoviridae, Badnavirus) (Table 1). Ten viruses had been already reported, frequently infecting grapevines in Brazil (GLRaV-2, −3, −4, −4 strain 5, GVA, GVB, GVD, GRSPaV, GFkV, GRVFV) (Basso et al 2017;Catarino et al 2015), three had not been previously reported in this country (GCSV, GRGV and GVCV) and one had only recently been identified (GSyV-1) (Moura et al 2017). It is interesting to highlight the association of some viruses with wild grapevine genotypes (Table 1).…”

“…There are few studies of diversity and genetic variability of Brazilian isolates of grape viruses. Determining which species are prevalent in specific regions, as well as performing genetic variability studies are important steps to understand how pathogens evolve and change, predicting epidemics and recommending control measures (Moura et al 2017). Based on genetic variability studies it is also possible to develop more consistent tools (such as specific primers) for diagnosis of plant viruses, by revealing more conserved regions in the genome of a particular viruses.…”