Eliezer R. Souto

Biological and molecular characterization of an isolate of Tobacco streak virus obtained from soybeans in Brazil

ABSTRACT A virus was isolated from soybean ( Glycine max ) plants with symptoms of dwarfing and bud blight in WenceslauBraz County, Parana, Brazil. The host range and properties resembled those of Tobacco streak virus (TSV). The purifiedvirus showed three peaks in a frozen sucrose gradient. Antiserum was produced and the virus was serologically related toTSV. Electron microscopy detected 28 nm spherical particles. Coat protein (CP) had a Mr of 29.880 Da. A fragment of 1028nt was amplified, cloned and sequenced. One open reading frame with 717 nt was identified and associated to the CP. TheCP gene shared 83% identity with the sequence of TSV CP from white clover ( Trifolium repens ) (GenBank CAA25133).This is the first report of the biological and molecular characterization of TSV isolated from soybeans. It is proposed thatthis isolate be considered a strain of TSV named TSV-BR. Additional keywords : nucleotide sequence, Elisa, host range. RESUMOCaracterizacao biologica e molecular de um isolado de

Effect of crop rotation on specialization and genetic diversity of Macrophomina phaseolina

Charcoal rot, caused by Macrophomina phaseolina, is one of the most important diseases of soybean. Genetic variability among soybean isolates has been observed but the effect of host specialization on genetic variability has not been reported. In this work, isolates from soybean, corn and sunflower were evaluated based on cross inoculations and number of microsclerotia/g of roots. The highest and lowest significant (P<0.005) production of microsclerotia was obtained in soybean (863 microsclerotia/g of roots) and sunflower (578 microsclerotia/g of roots), respectively, regardless of the origin of the isolate. Additionally, the effect of a 20-year crop rotation on genetic variability based on RAPD was studied. Eighty-nine isolates from five populations were obtained from soil samples under four crop rotation systems and an uncropped soil. Seven clusters were obtained considering a similarity level of 85%. Analysis indicated that M. phaseolina is a highly diverse species and also revealed a strong effect of the rotation system on genetic diversity. AMOVA was conducted for the RAPD data. From the total genetic variability, 21% (P<0.0001) could be explained by the differences between populations while 79% could be explained by differences within populations ( Φst = 0.2110; P<0.0001). The mean coefficient of gene differentiation (GST) estimated among the five populations indicated 27% of differentiation between populations similar to the AMOVA results where Φst= 0.2110. Total gene diversity estimated indicated high levels of variability (HT =0.3484). Results suggest that genetic differentiation of M. phaseolina can be altered by crop rotation.

Detection and identification of TMV infecting tomato under protected cultivation in Paraná State

During an inspection in plastic houses in Sapopema, Parana, 90% of tomato plants showed leaf abnormalities, probably associated with herbicide toxity. However, virus like symptoms developed in selected hosts after mechanical inoculatation. RT-PCR reactions using primers for an internal region within the movement protein gene of TMV and ToMV resulted in the amplification of a 409 bp cDNA fragment only by TMV primers. Deduced amino acids showed 100% identity when compared to TMV movement protein and 94% with ToMV. The RT-PCR protocol was efficient for quick and conclusive determination of virus species. The virus was purified and a polyclonal antiserum was raised for future surveys in tomato crops of Parana. The partial genomic sequence obtained for TMV-Sapopema has been deposited under the accession number DQ173945, which is the first partial genomic sequence of an isolate of TMV from Brazil in the GenBank, and the first tomato virus isolate from Parana to have some of its biological and molecular properties determined.

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.

Resistência de genótipos de videira à ferrugem

O objetivo deste trabalho foi avaliar a resistencia a ferrugem causada pelo fungo Phakopsora euvitis, em genotipos de videira, e a efi ciencia da infeccao de acordo com a idade das folhas e concentracao de inoculo. A avaliacao foi feita em 15 genotipos, com base nos seguintes componentes de resistencia: numero de pustulas (uredios) por cm2; tamanho das pustulas; numero de esporos produzidos por pustula; e periodo latente. Os componentes de resistencia, com excecao do periodo latente, apresentaram grande variacao quantitativa. A analise multivariada desses componentes diferenciou cinco grupos de genotipos. Os genotipos mais resistentes foram as cultivares porta-enxertos IAC313, IAC572 e IAC766, em que a efi ciencia da infeccao foi baixa, com pustulas menores e menor producao de urediniosporos, alem de reacao de hipersensibilidade no tecido em torno das pustulas. O grau de infeccao aumenta com o aumento na concentracao do inoculo. A ferrugem coloniza, indiscriminadamente, folhas jovens e folhas maduras de videira.

Immunocapture-RT-PCR detection of Cassava common mosaic virus in cassava obtained from meristem-tip culture in Paraná state

A virus survey conducted in the northwest region of Parana, the main cassava-producing region of that state, showed Cassava common mosaic virus (CsCMV) to be widespread, infecting more than 90% of plants from all cassava cultivars. A CsCMV isolate was purified and used to raise a high-titer (1/1.000) polyclonal antiserum for indexing plants produced from meristem-tip culture, using PTA-ELISA. From an initial production of 110 plants of cultivar Olho Junto, 31 remained infected as indicated by PTA-ELISA. To improve the sensitivity of virus detection, an immunocapture-RT-PCR (IC-RT-PCR) protocol was established. Virus-specific IgG, purified and combined with a primer set for the genus Potexvirus, could readily detect CsCMV in cassava crude leaf extracts. IC-RT-PCR products of 750 bp were amplified from six out of 35 plants previously tested as virus-negative by PTA-ELISA. Sequence analysis of cloned IC-RT-PCR products confirmed they were part of the CsCMV replicase gene, indicating that the virus was still present after thermotherapy and meristem-tip culture. PTA-ELISA enabled initial screening of virus-positive cassava, reducing the number of plants to be further analyzed by IC-RT-PCR. Though CsCMV has been considered of minor importance, its widespread nature, as noticed in Parana, indicates the need for adoption of effective control measures.

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.

Two distinct 16SrIII phytoplasma subgroups are associated with shoot proliferation in Vernonia brasiliana, a wild species inhabiting the Brazilian savanna

Vernonia brasiliana is a wild species frequently found in Brazilian savanna areas, also known as “cerrado” areas. In pasture fields, shrubs were observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, mild leaf chlorosis and deformed leaves. Molecular detection revealed the presence of phytoplasmas in approximately 80% of the symptomatic plants. Identification by computer-simulated RFLP allowed the classification of phytoplasma strains as representatives of ‘Candidatus Phytoplasma pruni’, taxonomic subgroups 16SrIII-B and 16SrIII-J. Moreover, phylogenetic analysis evidenced perfect agreement with virtual RFLP. Both phytoplasmas found in diseased vernonia plants have been reported in diverse commercial species cultivated in Brazil, including many vegetables and an extensive number of crops. These findings identify V. brasiliana as a new host of phytoplasmas affiliated with the subgroups 16SrIII-B and 16SrIII-J and suggest this species as a probable reservoir and possible inoculum source of phytoplasmas.

Detecção do Southern bean mosaic virus no Paraná, e separação do Bean rugose mosaic virus em feijoeiro

Em lavouras de feijoeiro (Phaseolus vulgaris) da cultivar Carioca Comum, no municipio de Londrina, Estado do Parana, foram encontradas plantas com sintomas de necrose da haste, mosaico clorotico leve e porte reduzido, semelhantes aos sintomas causados por infeccao viral. Exames de microscopia eletronica revelaram a presenca de particulas isometricas. Em testes de imunodifusao dupla em gel de agar os extratos foliares de plantas infetadas reagiram positivamente com anti-soro especifico para o Southern bean mosaic virus (SBMV). O virus foi purificado e a massa molecular de sua proteina capsidial foi estimada em 30 kDa, valor esperado para proteinas do capsideo de virus do genero Sobemovirus. A gama de hospedeiras do SBMV isolado no Parana foi restrita ao feijoeiro e a algumas cultivares de soja (Glycine max). A separacao de dois virus isometricos comuns em infeccoes mistas no feijoeiro foi possivel atraves da reacao de imunidade ao SBMV apresentada por Crotalaria sp, Chenopodium quinoa e Mucuna deeringiana, e da reacao de susceptibilidade dessas mesmas hospedeiras ao Bean rugose mosaic virus (BRMV).

Análise molecular de segmento do RNA2 de comovirus isolados de soja no estado do Paraná

In bean (Phaseolus vulgaris) producing areas of Parana State, Bean rugose mosaic virus (BRMV) has been found in mixed infections with Bean golden mosaic virus (BGMV) increasing the severity of symptoms and crop losses. The association of Cowpea severe mosaic virus (CPSMV) with bud blight symptoms in soybean (Glycine max) plants was recently observed in Parana for the first time. In this work RT-PCR products of 600 bp of BRMV-PR RNA2 and 594 bp of CPSMV-PR RNA2 were cloned and sequenced. The amino acid sequences coded by the RNA2 segment from these isolates were compared with sequences of the same RNA2 genomic region of six comovirus species from the GenBank. The CPSMV-PR showed 85% homology with the CPSMV RNA2 from the GenBank. Moreover, the BRMV-PR isolate showed 39 and 44% homology with CPSMV and Bean pod mottle virus (BPMV), respectively. This work shows, for the first time, partial sequencing data for BRMV. This data may be helpful for the complete molecular characterization of this virus and for establishing strategies to obtain virus resistant plants.