Avaliação da variabilidade do Grapevine leafroll-associated virus 1 e 3 por análise de seqüências de nucleotídeos e polimorfismo conformacional de fita simples

As principais espécies de vírus envolvidas na etiologia do enrolamento da folha da videira (Vitis spp.) são Grapevine leafroll-associated virus 1 e 3 (GLRaV-1 e -3). Neste estudo da variabilidade desses vírus, foram amplificados dois fragmentos de DNA (396 bp do GLRaV-1 e 602 bp do GLRaV-3) por RT-PCR, a partir de RNA total extraído de nervuras e pecíolos de videiras infetadas, utilizando-se dois pares de oligonucleotídeos. Os DNAs amplificados foram clonados e reamplificados, a partir dos clones recombinantes, e comparados quanto às diferenças conformacionais das fitas simples desnaturadas (SSCP). Foram observados dois padrões distintos de perfis eletroforéticos para cada vírus, tendo sido seqüenciado pelo menos um clone viral correspondente a cada padrão. As duas seqüências de nucleotídeos obtidas para o GLRaV-1 apresentaram maior homologia (79,8% e 87,4%) com um isolado australiano e as duas seqüências relativas ao GLRaV-3 exibiram maior homologia (75,1% e 81,8%) com um isolado norte-americano. Os resultados demonstraram a ocorrência de seqüências variantes destes vírus nas videiras analisadas.

Resistência de cultivares e linhagens de tomateiro a Tomato chlorotic spot virus e a Potato virus Y

Linhagens avançadas do programa de melhoramento do tomateiro (Lycopersicon esculentum) do IAC foram avaliadas em condições de campo em Campinas (SP) para resistência a tospovírus e a potyvírus, nos anos agrícolas 2002/2003 e 2003/2004, respectivamente. No primeiro ano, a única espécie de tospovírus que ocorreu na área experimental foi Tomato chlorotic spot virus (TCSV). As sete linhagens do grupo IAC exibiram baixa porcentagem de plantas sintomáticas em duas avaliações, com médias abaixo de 28%; as cultivares testadas mostraram-se altamente suscetíveis, com médias acima de 85%, à exceção de 'Franco', que apresentou cerca de 55% de infecção. No segundo experimento, conduzido em 2003/2004, dez linhagens do grupo IAC foram comparadas com cinco cultivares de polinização aberta e híbridos F1, além do acesso LA-444-1 de L. peruvianum. Nesse experimento, por meio de testes biológicos e sorológicos, verificou-se ocorrência generalizada de Potato virus Y (PVY). Foi determinado o percentual de plantas com sintomas e avaliada a intensidade dos sintomas mediante uso de escala de notas. Com base nos dois critérios, verificou-se que LA-444-1 apresenta alta resistência a PVY, que 'Tyrade' exibe comportamento intermediário, enquanto todos os demais genótipos demonstram alta suscetibilidade ao vírus. O comportamento dos genótipos avaliados neste trabalho mostra a necessidade de se considerar, nos programas de melhoramento do tomateiro, a introgressão de fatores de resistência não só a vírus de importância atual nas regiões produtoras, como geminivírus, mas também a outros vírus potencialmente nocivos à cultura, como tospovírus e potyvírus.

Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the São Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong Nº2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

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.

Effect of Potato virus X on total phenol and alkaloid contents in Datura stramonium leaves

The present paper reports results of the effect of Potato virus X (PVX) on the contents of total phenols and alkaloids in leaves of Datura stramonium. A significant decrease in the contents of phenols and alkaloids was observed in leaves inoculated with PVX (X-I). However, there was an increase in the percentage of phenols in leaves rubbed with phosphate buffer (C1-I) and in leaves from the nodes immediately above, possibly induced by mechanical injury. Gas chromatography/mass spectroscopy revealed amounts of scopolamine in samples submitted to all treatments, except X-I, in which the amount of this alkaloid was low. High amounts of an unidentified compound (molecular ion m/z 302 and a prominent peak at m/z 129) were noted in extracts from leaves X-I, C1-I and leaves from the nodes immediately above the leaves inoculated with PVX. It is suggested that the synthesis and accumulation of the unidentified compound is a result of stress from mechanical injury and virus inoculation.

Detecção do Grapevine leafroll-associated virus 5 no Estado de São Paulo

O enrolamento da folha da videira ("grapevine leafroll") é uma doença atribuída a pelo menos nove vírus sorologicamente distintos, Grapevine leafroll-associated viruses 1 a 9 e designados GLRaV-1 a GLRaV-9. No Brasil, já é conhecida a existência do GLRaV-1, GLRaV-2, GLRaV-3 e GLRaV-6. Neste trabalho, foi demonstrada a ocorrência do GLRaV-5 em amostras de videiras cultivadas no Estado de São Paulo, mediante teste de Biotina-ELISA. O vírus foi detectado com baixa incidência nas cultivares avaliadas, exceto na 'Cardinal', que apresentou 100% de infecção. Este é o primeiro relato da ocorrência do GLRaV-5 no Brasil.

Solanum americanum: reservoir for Potato virus Y and Cucumber mosaic virus in sweet pepper crops

Weeds can act as important reservoirs for viruses. Solanum americanum (Black nightshade) is a common weed in Brazil and samples showing mosaic were collected from sweet pepper crops to verify the presence of viruses. One sample showed mixed infection between Cucumber mosaic virus (CMV) and Potato virus Y (PVY) and one sample showed simple infection by PVY. Both virus species were transmitted by plant extract and caused mosaic in tomato (Solanum lycopersicum cv. Santa Clara), sweet pepper (Capsicum annuum cv. Magda), Nicotiana benthamiana and N. tabaccum TNN, and local lesions on Chenopodium quinoa, C. murale and C. amaranticolor. The coat protein sequences for CMV and PVY found in S. americanum are phylogenetically more related to isolates from tomato. We conclude that S. americanum can act as a reservoir for different viruses during and between sweet pepper crop seasons.

Detecção do Grapevine leafroll-associated virus 5 no Estado de São Paulo

O enrolamento da folha da videira (grapevine leafroll) é uma doença atribuída a pelo menos nove vírus sorologicamente distintos, Grapevine leafroll-associated viruses 1 a 9 e designados GLRaV-1 a GLRaV-9. No Brasil, já é conhecida a existência do GLRaV-1, GLRaV-2, GLRaV-3 e GLRaV-6. Neste trabalho, foi demonstrada a ocorrência do GLRaV-5 em amostras de videiras cultivadas no Estado de São Paulo, mediante teste de Biotina-ELISA. O vírus foi detectado com baixa incidência nas cultivares avaliadas, exceto na 'Cardinal', que apresentou 100% de infecção. Este é o primeiro relato da ocorrência do GLRaV-5 no Brasil.

Solanum americanum: reservoir for Potato virus Y and Cucumber mosaic virus in sweet pepper crops

Weeds can act as important reservoirs for viruses. Solanum americanum (Black nightshade) is a common weed in Brazil and samples showing mosaic were collected from sweet pepper crops to verify the presence of viruses. One sample showed mixed infection between Cucumber mosaic virus (CMV) and Potato virus Y (PVY) and one sample showed simple infection by PVY. Both virus species were transmitted by plant extract and caused mosaic in tomato (Solanum lycopersicum cv. Santa Clara), sweet pepper (Capsicum annuum cv. Magda), Nicotiana benthamiana and N. tabaccum TNN, and local lesions on Chenopodium quinoa, C. murale and C. amaranticolor. The coat protein sequences for CMV and PVY found in S. americanum are phylogenetically more related to isolates from tomato. We conclude that S. americanum can act as a reservoir for different viruses during and between sweet pepper crop seasons.

Insecticidal Activity of the Granulosis Virus in Combination with Neem Products and Talc Powder Against the Potato Tuberworm Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

The potato tuberworm Phthorimaea operculella (Zeller) is an important agricultural pest that causes significant economic losses to potato growers worldwide. The addition of an effective method of biological control for the potato tuberworm is greatly needed, and is currently unavailable in Brazil. The granulosis virus (Baculoviridae) is a promising biological control agent to protect post-harvest potatoes and in storage from the potato tuberworm. However, the control measure must be economically feasible. Liquid suspensions of a granulosis virus applied alone or in mixture with two commercial neem oil-based products (DalNeem (TM) and NeemAzal (TM)), and a dry powder formulation of viral granules were evaluated for control of potato tuberworm larvae by treating potato tubers under laboratory conditions. High larval mortality (86.7%) was achieved when DalNeem and virus were applied together at 4 mg of azadirachtin/L and 10(4) occlusion bodies (OBs)/mL, respectively. This combination resulted in a parts per thousand yen50% efficacy in relation to their counterparts alone. Conversely, NeemAzal did not enhance virus effectiveness against larvae of the potato tuberworm. The talc-based virus formulation was used for dusting seed tubers at different concentrations and resulted in 100% larval mortality at 5 x 10(8) OBs/g. Formulated and unformulated virus provided 50% mortality at 166 OBs/g and at 5.0 x 10(5) OBs/mL, respectively. As a result, talc-based virus formulation had a better control efficiency on potato tuberworm than the aqueous virus suspension. The granulosis virus combined with DalNeem at low rates or formulated with talc powder is a viable option to control the potato tuberworm under storage conditions.

Transcriptional changes and oxidative stress associated with the synergistic interaction between potato virus X and potato virus Y and their relationship with symptom expression.

Many virus diseases of economic importance to agriculture result from mixtures of different pathogens invading the host at a given time. This contrasts with the relatively scarce studies available on the molecular events associated with virus---host interactions in mixed infections. Compared with single infections, co-infection of Nicotiana benthamiana with Potato virus X (PVX) and Potato virus Y (PVY) resulted in increased systemic symptoms (synergism) that led to necrosis of the newly emerging leaves and death of the plant. A comparative transcriptional analysis was undertaken to identify quantitative and qualitative differences in gene expression during this synergistic infection and correlate these changes with the severe symptoms it caused. Global transcription profiles of doubly infected leaves were compared with those from singly infected leaves using gene ontology enrichment analysis and metabolic pathway annotator software. Functional gene categories altered by the double infection comprise suites of genes regulated coordinately, which are associated with chloroplast functions (downregulated), protein synthesis and degradation (upregulated), carbohydrate metabolism (upregulated), and response to biotic stimulus and stress (upregulated). The expressions of reactive oxygen species?generating enzymes as well as several mitogen-activated protein kinases were also significantly induced. Accordingly, synergistic infection induced a severe oxidative stress in N. benthamiana leaves, as judged by increases in lipid peroxidation and by the generation of superoxide radicals in chloroplasts, which correlated with the misregulation of antioxidative genes in microarray data. Interestingly, expression of genes encoding oxylipin biosynthesis was uniquely upregulated by the synergistic infection. Virus-induced gene silencing of ?-dioxygenase1 delayed cell death during PVX?PVY infection.

Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.

Divinyl ether fatty acid synthesis in late blight-diseased potato leaves.

We conducted a study of the patterns and dynamics of oxidized fatty acid derivatives (oxylipins) in potato leaves infected with the late-blight pathogen Phytophthora infestans. Two 18-carbon divinyl ether fatty acids, colneleic acid and colnelenic acid, accumulated during disease development. To date, there are no reports that such compounds have been detected in higher plants. The divinyl ether fatty acids accumulate more rapidly in potato cultivar Matilda (a cultivar with increased resistance to late blight) than in cultivar Bintje, a susceptible cultivar. Colnelenic acid reached levels of up to approximately 24 nmol (7 microgram) per g fresh weight of tissue in infected leaves. By contrast, levels of members of the jasmonic acid family did not change significantly during pathogenesis. The divinyl ethers also accumulated during the incompatible interaction of tobacco with tobacco mosaic virus. Colneleic and colnelenic acids were found to be inhibitory to P. infestans, suggesting a function in plant defense for divinyl ethers, which are unstable compounds rarely encountered in biological systems.