Osmar Nickel

Sequence analysis of the capsid protein gene of an isolate of Apple stem grooving virus, and its survey in Southern Brazil

Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Development of virus resistant transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus

Translatable and nontranslatable versions of the coat protein (cp) gene of a Papaya ringspot virus (PRSV) isolate collected in the state of Bahia, Brazil, were engineered for expression in Sunrise and Sunset Solo varieties of papaya (Carica papaya). The biolistic system was used to transform secondary somatic embryo cultures derived from immature zygotic embryos. Fifty-four transgenic lines, 26 translatable and 28 nontranslatable gene versions, were regenerated, with a transformation efficiency of 2.7%. Inoculation of cloned R0 plants with PRSV BR, PRSV HA or PRSV TH, Brazilian, Hawaiian and Thai isolates, respectively, revealed lines with mono-, double-, and triple-resistance. After molecular analysis and a preliminary agronomic evaluation, 13 R1 and R2 populations were incorporated into the papaya-breeding program at Embrapa Cassava and Tropical Fruits, in Cruz das Almas, Bahia, BrazilVersoes traduziveis e nao traduziveis do gene da capa proteica (cp) de um isolado de Papaya ringspot virus (PRSV) coletado no Estado da Bahia, Brasil, foram produzidas para expressao nas variedades Sunrise e Sunset Solo de mamoeiro (Carica papaya). O sistema de biobalistica foi utilizado para transformar embrioes somaticos secundarios derivados de embrioes zigoticos imaturos. Cinquenta e quatro linhas transgenicas, sendo 26 contendo versoes traduziveis e 28 contendo versoes nao traduziveis do gene cp foram regeneradas, o que resultou em 2,7% de eficiencia de transformacao, quando considerado o numero de linhas transgenicas obtidas por embriao zigotico imaturo excisado. Desafios de plantas R0 com PRSV BR, PRSV HA ou PRSV TH, respectivamente isolado brasileiro, havaiano e tailandes, revelaram linhas com resistencia a um, dois e tres isolados de PRSV. Apos analises moleculares e avaliacao agronomica preliminar, 13 populacoes R1 e R2 de mamoeiros transgenicos foram incorporadas ao programa de melhoramento genetico da Embrapa Mandioca e Fruticultura, em Cruz das Almas, Bahia, Brasil.

Fisiologia foliar e qualidade enológica da uva em videiras infectadas por vírus

Leaf physiology and enologic grape quality of virus-infected plants Viruses may induce metabolic and structural disarray in plant cells to varying degrees depending on viral species and plant susceptibility. With this focus, grapevine plants (Vitis vinifera) cvs. Cabernet Franc and Cabernet Sauvignon, symptomless and showing symptoms of virus-infection, in two commercial vineyards were comparatively analyzed. The parameters were: 1. photosynthetic potential (light-saturated rate of photosynthesis, saturating light intensities, light compensation point, dark respiration rate, apparent quantum yield, chlorophyll a and b); 2. foliar carbon metabolism (total soluble sugars and starch), and 3. enologic quality of the produced grapes (total soluble solids - °Brix, density, pH and titerable total acidity in the must; total color intensity and total polyphenols in berry skins) were recorded. In symptomatic plants Grapevine leafroll-associated virus 2 (GLRaV-2) and Rupestris stem pitting-associated virus (RSPaV) have been detected by ELISA carried out for six viruses. The virus infections induced significant reductions in chlorophyll content and in photosynthetic potential of both cultivars. Leaves of infected plants also showed significant accumulation of carbohydrates, suggesting a blockage of carbon transport out of these tissues. Concerning the enologic quality and based on technological parameters of the must (°Brix, density, titerable total acidity and polyphenols), grapes of infected plants showed at harvest a significantly lower level of maturation. These viruses directly affect the productive capacity as well as the quality of the grapes produced by these cultivars.

Detecção e identificação molecular de vírus associados a videiras sintomáticas e assintomáticas

The vegetative propagation of grapevine facilitates multiple viral infections, with different symptoms which vary according to combinations of cultivar or host species with viral species. The aims of this research were to detect and identify the viral species infecting two grapevine species/cultivars: one symptomatic and one symptomless. DsRNA from both samples was assayed by RT-PCR using 17 pairs of specific primers for detection of the Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV) and Grapevine leafroll-associated virus 1-4 (GLRaV-1 to -4), besides three degenerate primer pairs. For each primer pair at least one amplicon was cloned and sequenced. Symtomatic and symptomless plants were multiple infected by RSPaV, GLRaV-2 and/or GLRaV-3. The nucleotide sequences of seven isolates of RSPaV, three of GLRaV-2 and two of GLRaV-3 showed identities higher than 90% with the homologous viral species and allowed to identify possible viral strains in infected samples. These results highlight the necessity of viral diagnosis based on specific assays to determine grapevine sanitary status.

Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil

ABSTRACT An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca andV. vinifera grapevines on the indicator LN33,was transmitted mechanically to several Nicotiana species. The viruswas partially purified from N. cavicola and the coat protein estimatedat 23 kDa by SDS-PAGE. In negatively stained leaf extracts ofexperimentally inoculated N. cavicola and N. occidentalis , flexuousparticles with cross banding were observed, predominantlymeasuring 750-770 x 12 nm, with a modal length of 760 nm.Decoration indicated a clear, positive reaction against AS-GVB. InDAS-ELISA, GVB was detected in N. cavicola and grapevineextracts, and Western blots showed homologous and cross reactionof GVB and GVA antisera with GVB coat protein. Using specificprimers for GVB, a fragment of 594 bp, comprising the coat proteingene coding for 197 amino acids, was amplified by RT-PCR withviral RNA extracted from GVB-infected N. occidentalis . Thenucleotide and the deduced amino acid sequences of the coat proteingene showed high identities with Italian and Japanese isolates ofGVB.

Simultaneous detection of Brazilian isolates of grapevine viruses by TaqMan real-time RT-PCR

The aim of this work was to evaluate the efficiency of real-time RT-PCR for detection of different isolates of ten important virus species that infect grapevines in Brazil: Grapevine leafroll-associated virus (GLRaV-1, -2, -3 and -5), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fleck virus (GFkV) and Grapevine fanleaf virus (GFLV). The reactions consisted of individual (simplex) and simultaneous (duplex) virus detections. Thirty six grapevine accessions, regenerated after thermotherapy and tissue culture treatments, have been analysed. All the above-mentioned viruses were sensitively detected in simplex reactions in samples infected with different virus isolates. Specifically to GLRaV-1 it was necessary to mix reagents refered by different sources to achieve the amplification. GVA, GRSPaV, GLRaV-2 and GLRaV-3 combined with GVB, GFLV, GFkV, GVD and GLRaV-5 were accurately detected in duplex trials. It was shown, that real-time RT-PCR (TaqMan) is able to efficiently detect different local virus species and isolates.

Coat protein gene variability of three viral species infecting grapevines in Brazil

The purpose of this study was to evaluate the variability of three viruses (Rupestris stem pitting-associated virus - RSPaV, Grapevine leafroll-associated virus 2 - GLRaV-2 and Grapevine fanleaf virus - GFLV) infecting grapevines in Brazil, through molecular characterization of the coat protein (CP) gene. DNA fragments were amplified comprising the complete CP genes of nine isolates of RSPaV (780 bp), six of GLRaV-2 (597 bp) and three of GFLV (1515 bp) by RT-PCR, using specific primers for each viral species. The amplified DNA fragments were cloned and sequenced. RSPaV isolates were clustered into four groups by phylogenetic analysis of the nucleotide (nt) sequences of the CP gene, showing identity values ranging from 81 to 99%. For GLRaV-2, two groups were defined from nt sequences, with identity values ranging from 88 to 99% and for GFLV, two groups were defined with identity values ranging from 89 to 98%. The isolates of each viral species studied here were detected by non-radioactive probes labeled with digoxigenin, allowing unambiguous identification of infected samples, independent of the isolate used as template for probe synthesis.

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

The application of high-throughput sequencing technologies (HTS) enables the recovery of many nucleotide sequence fragments from diseased plants and may help in pathogen identification. This study was designed to identify viruses infecting 15 grapevine (Vitis spp.) samples collected from experimental fields and vine collections and assess the genetic variability of the identified viruses. The virus-enriched dsRNAs were extracted from bark scrapings and sequenced using an Illumina platform. The paired-end reads were analyzed, assembled contigs were generated and identified as related to viruses. Contigs of 14 viruses have been identified, some of them covering large extensions of viral genomes or resulting in assembly of near-complete or complete genomes. Grapevine virus infections are usually mixed and the HTS assays were suitable to identify ten viruses already reported that traditionally infect grapevines in Brazil, one that has been recently identified (Grapevine Syrah virus 1) and others (Grapevine Cabernet Sauvignon reovirus, Grapevine Red Globe virus and Grapevine vein clearing virus) not previously reported in this country. Nucleotide identities among Brazilian isolates identified by HTS and homologous grapevine virus sequences in GenBank were high, ranging from 77% to 99%. Genetic variability analysis of viral sequences obtained by HTS and sequences available in GenBank indicated that the coding regions in the different viral species are under purifying selection, and that recombination events occurred in the majority of the viral species analyzed. The coat protein genes, generally, had lower genetic variability than the replicase and movement protein genes.

Detection and partial molecular characterization of Grapevine fleck virus, Grapevine virus D, Grapevine leafroll-associated virus -5 and -6 infecting grapevines in Brazil

Grapevine fleck, rugose wood and leafroll are three grapevine viral diseases whose causal agents (or associated viruses) respectively are Grapevine fleck virus (GFkV), Grapevine virus D (GVD) and Grapevine leafroll-associated virus 5 and 6 (GLRaV-5 and -6). The objective of this work was to perform a partial molecular characterization of local isolates of these four viral species that infect grapevines. The nucleotide and deduced amino acid sequences of complete genes of the coat protein (CP) (of GFkV), the CP and the RNA binding protein (of GVD), the CP and the partial hHSP70 gene (of GLRaV-5) and the partial hHSP70 gene (of GLRaV-6) were aligned and compared in silico with other isolates. These data extend the available information about Brazilian isolates of GFkV, GLRaV-5 and -6, and reports for the first time the GVD occurrence in Brazil.