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Search Results: 1 - 10 of 492301 matches for " Thor V.M.;Flores "
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Citrus exocortis viroid and Hop Stunt viroid Doubly infecting grapevines in Brazil
Eiras, Marcelo;Targon, Maria Luisa P.N.;Fajardo, Thor V.M.;Flores, Ricardo;Kitajima, Elliot W.;
Fitopatologia Brasileira , 2006, DOI: 10.1590/S0100-41582006000500002
Abstract: viroids, non-protein-coding small (246-401 nt) circular single-stranded rnas with autonomous replication, are currently classified into two families. within the family pospiviroidae, citrus exocortis viroid (cevd) belongs to the genus pospiviroid while hop stunt viroid (hsvd) is the single member of the genus hostuviroid. these pathogens are distributed worldwide and infect a large number of hosts. in brazil, isolates of cevd and hsvd have been detected in both citrus and grapevine. to characterize and study the genetic variability of these viroids, total rna from leaves of grapevine vitis vinifera 'cabernet sauvignon' and v. labrusca 'niagara rosada' from bento gon?alves, rs, was used as a template for rt-pcr amplification with specific primers for the five viroids described infecting grapevines [hsvd, cevd, grapevine yellow speckle viroid 1 (gysvd-1), grapevine yellow speckle viroid 2 (gysvd-2) and australian grapevine viroid (agvd)]. leaf samples of citrus medica infected with cevd from s?o paulo were also analyzed. the resulting products were separated by agarose gel electrophoresis and dna fragments of the expected size were eluted, cloned and sequenced. the grapevine samples analyzed were doubly infected by cevd and hsvd. a phylogenetic analysis showed that the brazilian grapevine hsvd variants clustered with other grapevine hsvd variants, forming a specific group separated from citrus variants, whereas the brazilian cevd variants clustered with other citrus and grapevine variants.
Detec??o e caracteriza??o biológica e molecular de Rupestris stem-pitting associated virus e seu efeito na fotossíntese de videiras
Fajardo, Thor V.M.;Eiras, Marcelo;Santos, Henrique P.;Nickel, Osmar;Kuhn, Gilmar B.;
Fitopatologia Brasileira , 2004, DOI: 10.1590/S0100-41582004000200016
Abstract: the rupestris stem-pitting associated virus (rspav) is reported to cause stem pitting disease in grapevines (vitis spp.). this work studies an rspav-isolate found infecting grapevine cv. c. franc in the state of rio grande do sul, brazil. this isolate was detected biologically, by grafting on rupestris du lot grapevine indicator, in 26.2% of the analysed samples. the partial replicase gene sequence of the southern brazilian rspav isolate, with 831 nucleotides amplified by rt-pcr and 276 deduced amino acids, showed high identities of nucleotides (98.1%) and deduced amino acids (99.6%), with two rspav north american isolates. the rspav studied showed low homology (37-41%) with other members of the genus foveavirus. most of the virus-free rootstocks grafted with rspav infected scions showed photosynthetic potential reduction (2.68 to 5.12 times) and dark respiratory rate increase when compared to healthy ones, emphasizing the potential negative impact of this virus on grapevine production.
Avalia??o da variabilidade do Grapevine leafroll-associated virus 1 e 3 por análise de seqüências de nucleotídeos e polimorfismo conformacional de fita simples
Fajardo, Thor V.M.;Eiras, Marcelo;Schenato, Paula G.;Nickel, Osmar;Kuhn, Gilmar B.;
Fitopatologia Brasileira , 2005, DOI: 10.1590/S0100-41582005000200013
Abstract: the main viral species associated with grapevine (vitis spp.) leafroll etiology are grapevine leafroll-associated virus 1 and 3 (glrav-1 and -3). to partially evaluate the variability of these viruses, two dna fragments (396 bp of glrav-1 and 602 bp of glrav-3) were amplified by rt-pcr using two sets of primers after total rna extraction from veins and petioles of infected grapevines. the amplified dna fragments were cloned and the reamplified viral dnas, obtained from the recombinant clones, were studied with regard to denatured single-strand dna conformational differences. two different electrophoretic profiles were observed for each virus species. at least one viral clone representing each mobility profile was sequenced. the two nucleotide sequences obtained for glrav-1 showed the highest homologies (79,8% and 87,4%) with an australian isolate, whereas the two nucleotide sequences of glrav-3 exhibited the highest homologies (75,1% and 81,8%) with a north american isolate. these results demonstrated the occurrence of glrav-1 and -3 sequence variants infecting the analysed grapevines.
Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies
Fajardo, Thor V.M.;Barros, Danielle R.;Nickel, Osmar;Kuhn, Gilmar B.;Zerbini, F. Murilo;
Fitopatologia Brasileira , 2007, DOI: 10.1590/S0100-41582007000600007
Abstract: 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.
Sequence analysis of the capsid protein gene of an isolate of Apple stem grooving virus, and its survey in Southern Brazil
NICKEL, OSMAR;FAJARDO, THOR V.M.;JELKMANN, WILHELM;KUHN, GILMAR B.;
Fitopatologia Brasileira , 2001, DOI: 10.1590/S0100-41582001000300014
Abstract: 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%).
Detec??o de Closterovirus em videira e caracteriza??o parcial de um isolado do Grapevine leafroll-associated virus 3
FAJARDO, THOR V.M.;KUHN, GILMAR B.;EIRAS, MARCELO;NICKEL, OSMAR;
Fitopatologia Brasileira , 2002, DOI: 10.1590/S0100-41582002000100009
Abstract: grapevine (vitis spp.) leafroll is a disease caused by up to eight viruses, denominated grapevine leafroll-associated viruses (glrav- 1 to -8), which are serologically unrelated and associated with phloem tissue. in the present paper, glrav-1 and -3 were detected by das-elisa in 6.9 and 14.7% of the samples, respectively, and glrav-2, -5 and -7 were not detected in grapes. the analysed samples came from two of the most important grape growing regions in brazil (serra gaúcha and vale do s?o francisco). the glrav-3 was also identified by dot-elisa and western blot, associated with a probable coat protein subunit of approximately 36 kda. a fragment of 340 bp, comprising the 3' terminal of the viral polymerase gene, was pcr-amplified and sequenced. the nucleotide and deduced amino acid sequences of this isolate showed high homology, 95 and 97.1%, respectively, with another glrav-3 isolate (ny1).
Variabilidade do gene da proteína capsidial de três espécies virais que infectam videiras no Brasil
Radaelli, Paula;Fajardo, Thor V.M;Nickel, Osmar;Eiras, Marcelo;Pio-Ribeiro, Gilvan;
Tropical Plant Pathology , 2009, DOI: 10.1590/S1982-56762009000500002
Abstract: 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.
Indexa??o biológica múltipla e RT-PCR para detec??o de vírus latentes em macieiras
Silva, Fabio N.;Nickel, Osmar;Fajardo, Thor V.M.;Bogo, Amauri;
Tropical Plant Pathology , 2008, DOI: 10.1590/S1982-56762008000200011
Abstract: this study aimed to evaluate the suitability of a multiple biological indexing method of latent apple viruses. the method consisted of grafting five indicator species and two buds of the plant to be indexed on to one rootstock. results showed that the sensitivity and efficacy of the mbi is adequate for reliable detection of apple stem grooving virus, (asgv), apple stem pitting virus (aspv) and apple chlorotic leaf spot virus (aclsv) of mother plant stock in certification programs. sample analysis by rt-pcr confirmed the results of the diagnosis of aspv and asgv by multiple biological indexing. however, it did not detect aclsv diagnosed by ll-s5 in three out of nine analyzed accessions. the coat protein gene of one of the accessions studied was cloned and sequenced. the annealing sites of primers used in unsuccessful amplification attempts were compared with data of the genbank to explain the non-amplification of certain aclsv isolates.
Simultaneous detection of Brazilian isolates of grapevine viruses by TaqMan real-time RT-PCR
Carla R. Dubiela,Thor V.M. Fajardo,Eliezer R. Souto,Osmar Nickel
Tropical Plant Pathology , 2013,
Abstract: 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.
Detec o e caracteriza o molecular parcial do Grapevine fleck virus em videiras
Fajardo Thor V.M.,Eiras Marcelo,Schenato Paula G.,Nickel Osmar
Fitopatologia Brasileira , 2004,
Abstract:
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