Ocorrência de begomovírus de plantas de pimentão no Estado de São Paulo e comportamento de genótipos de Capsicum spp. ao tomato severe rugose virus e a Bemisia tabaci meam1

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. © 2008 Elsevier B.V. All rights reserved.

The evolutionary value of recombination is constrained by genome modularity

Genetic recombination is a fundamental evolutionary mechanism promoting biological adaptation. Using engineered recombinants of the small single-stranded DNA plant virus, Maize streak virus (MSV), we experimentally demonstrate that fragments of genetic material only function optimally if they reside within genomes similar to those in which they evolved. The degree of similarity necessary for optimal functionality is correlated with the complexity of intragenomic interaction networks within which genome fragments must function. There is a striking correlation between our experimental results and the types of MSV recombinants that are detectable in nature, indicating that obligatory maintenance of intragenome interaction networks strongly constrains the evolutionary value of recombination for this virus and probably for genomes in general.

Rapid host adaptation by extensive recombination

Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature. © 2009 SGM.

Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts

Background. Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur. Results. We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 × 10 -4substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Réunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift. Conclusion. The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts. © 2009 Harkins et al; licensee BioMed Central Ltd.

A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family

Background. We have characterised a new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass. ECSV represents a new genus-level geminivirus lineage, and has a mixture of features normally associated with other specific geminivirus genera. Results. Whereas the ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses, curtoviruses and topocuviruses, its Rep also contains what is apparently a canonical retinoblastoma related protein interaction motif such as that found in mastreviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene found in begomoviruses, a mastrevirus-like coat protein gene and two intergenic regions. Conclusion. Although it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses. In addition to inferring how the ancestral geminivirus genome may have looked, we use the discovery of ECSV to refine various hypotheses regarding the recombinant origins of the major geminivirus lineages. © 2009 Varsani et al; licensee BioMed Central Ltd.

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

Molecular characterisation of dicot-infecting mastreviruses from Australia

Monocotyledonous and dicotyledonous plant infecting mastreviruses threaten various agricultural systems throughout Africa, Eurasia and Australasia. In Australia three distinct mastrevirus species are known to infect dicotyledonous hosts such as chickpea, bean and tobacco. Amongst 34 new "dicot-infecting" mastrevirus full genome sequences obtained from these hosts we discovered one new species, four new strains, and various variants of previously described mastrevirus species. Besides providing additional support for the hypothesis that evolutionary processes operating during dicot-infecting mastrevirus evolution (such as patterns of pervasive homologous and non-homologous recombination, and strong purifying selection acting on all genes) have mostly mirrored those found in their monocot-infecting counterparts, we find that the Australian dicot-infecting viruses display patterns of phylogeographic clustering reminiscent of those displayed by monocot infecting mastrevirus species such as Panicum streak virus and Maize streak virus.

Genetic diversity of subgroup 1 ilarviruses from eastern Australia

This is the first report of the genetic diversity within ilarvirus subgroup 1 from eastern Australia. It supports the separation of tobacco streak virus (TSV) strains from parthenium (Parthenium hysterophorus) and crownbeard (Verbescina encelioides) based on serology and host specificity. It has confirmed one previously described strain of TSV as a member of the species Strawberry necrotic shock virus and another as a new subgroup 1 ilarvirus, ageratum latent virus (AgLV), from Ageratum houstonianum. A multiplex RT-PCR showed that the genetically distinct strains of TSV and AgLV were commonly found in symptomless infections in virus-specific alternative weed hosts growing over a wide geographical range in eastern Australia. TSV has been one of the most damaging viruses in Australian oilseed and pulse crops in recent years, and this study has provided the taxonomic knowledge essential for the development of control programs for these viruses. © 2013 Springer-Verlag Wien.

Manual de identificação de doenças de soja.

Doenças causadas por fungos: Antracnose (Colletotrichum truncatum), Cancro da haste (Diaporthe phaseolorum var. meridionalis e D. phaseolorum var. caulivora), Crestamento foliar de cercóspora e mancha púrpura (Cercospora kikuchii), Ferrugem (Phakopsora pachyrhizi e P. meibomiae), Mancha alvo e podridão radicular de corinéspora (Corynespora cassiicola), Mancha foliar de ascoquita (Ascochyta sojae), Mancha foliar de mirotécio (Myrothecium roridum), Mancha olho-de-rã (Cercospora sojina), Mancha parda (Septoria glycines), Mela ou requeima (Rhizoctonia solani AG1), Míldio (Peronospora manshurica), Tombamento e morte em reboleira de rizoctonia (Rhizoctonia solani), Tombamento e murcha de esclerócio (Sclerotium rolfsii), Oídio (Erysiphe diffusa), Podridão branca da haste (Sclerotinia sclerotiorum), Podridão de carvão da raiz (Macrophomina phaseolina), Podridão parda da haste (Cadophora gregata), Podridão radicular de roselínia (Rosellinia necatrix), Seca da haste e da vagem (Phomopsis spp.), Podridão radicular de fitóftora (Phytophthora sojae), Podridão vermelha da raiz (Fusarium spp.). Doenças causadas por bactérias: Crestamento bacteriano (Pseudomonas savastanoi pv. glycinea), Fogo Selvagem (Pseudomonas syringae pv. tabaci), Pústula bacteriana (Xanthomonas axonopodis pv. glycines). Doenças causadas por vírus: Mosaico cálico (Alfalfa Mosaic Virus - AMV), Mosqueado do feijão (Bean Pod Mottle Virus - BPMV), Mosaico comum da soja (Soybean Mosaic Virus - SMV), Necrose da haste (Cowpea Mild Mottle Virus - CPMMV), Queima do broto (Tobacco Streak Virus - TSV). Doenças causadas por nematóides: Nematóide de cisto (Heterodera glycines), Nematóides de galhas (Meloidogyne incognita e M. javanica), Nematóide das lesões (Pratylenchus spp.), Nematóide reniforme (Rotylenchulus reniformis). Estádios de desenvolvimento da soja.

Begomovírus infectando a cultura de pimentão no estado de São Paulo

Vírus do gênero Begomovirus são transmitidos por mosca-branca Bemisia tabaci G., e constituem um dos problemas fitossanitários sérios em diversas culturas. Plantas de pimentão coletadas em oito regiões do Estado de São Paulo, foram submetidas a extração de DNA total e PCR com primers universais e degenerados para begomovírus, que amplificam parte da região codificadora para a proteína capsicial. Os dados indicam a presença de begomovírus em pimentão nas cinco regiões coletadas. Análise das seqüências do DNA viral e análise filogenética revelaram identidade com dois begomovírus nativo da América. Tomato severe rugose virus - ToSRV (AY029750) e com Tomato yellow vein streak virus (ToYVSV, AY829113), espécies descritas infectando tomateiro no Brasil. A presença de begomovírus em pimentão foi verificada nas regiões de Alvinlândia, Ubirajara, Botucatu, Elias-Fausto, Paulínia, Mogi Guaçu, Paranapanema e Pirajú.

Diversidade genética de begomovírus em cultivos de tomateiro no Centro-Oeste Paulista

A diversidade genética de vírus pertencentes ao gênero Begomovirus em tomateiro (Lycopersicon esculentum Mill) foi analisada em regiões produtoras do Centro-Oeste paulista. No período de janeiro de 2003 a fevereiro de 2004, cento e sessenta e seis amostras de tomate foram coletadas e a presença de begomovírus observada em 60% das amostras, por PCR, utilizando-se oligonucleotídeos universais para o gênero Begomovirus. O sequenciamento direto do produto de PCR de 16 dessas amostras indicou a possível presença do Tomato severe rugose virus (ToSRV), Sida mottle virus (SiMoV-[BR]) e da espécie tentativa Tomato yellow vein streak virus (ToYVSV-[BR]). em duas amostras foi detectada uma possível nova espécie de begomovírus. A presença do ToSRV e do SiMoV ainda não havia sido verificada em tomateiro no estado de São Paulo. Estes resultados indicam a existência de diversidade de espécies de begomovírus infectando o tomateiro nesta região, servindo como um alerta para melhoristas que trabalham na busca de fontes de resistência a esse importante grupo de patógenos.

Análise da diversidade genética de Begomovírus em tomateiro (Lycopersicon esculentum Mill) no Centro-Oeste Paulista

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudos biológicos e moleculares de begomovirus infectando pimentão (Capsicum annuum) no Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)