Marcelo Eiras

Detection of three Allexivirus species infecting garlic in Brazil

Garlic viruses often occur in mixed infections under field conditions. In this study, garlic samples collected in three geographical areas of Brazil were tested by Dot-ELISA for the detection of allexiviruses using monoclonal specific antibodies to detect Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and a polyclonal antiserum able to detect the three virus species mentioned plus Garlic virus D (GarV-D). The detected viruses were biologically isolated by successive passages through Chenopodium quinoa. Reverse Transcriptase Polimerase Chain Reaction (RT-PCR) was performed using primers designed from specific regions of the coat protein genes of Japanese allexiviruses available in the Genetic Bank of National Center of Biotechnology Information (NCBI). By these procedures, individual garlic virus genomes were isolated and sequenced. The nucleotide and amino acid sequence analysis and the one with serological data revealed the presence of three distinct allexiviruses GarV-C, GarV-D and a recently described allexivirus, named Garlic mite-borne filamentous virus (GarMbFV), in Brazil.

RT-PCR and dot blot hybridization methods for a universal detection of tospoviruses.

Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.

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 Sao 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.

Citrus exocortis viroid and Hop Stunt viroid Doubly infecting grapevines in Brazil

Part of the Doctoral Thesis of the first author. ESALQ, Universidade de Sao Paulo, Piracicaba, SP. 2006.

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.

Resistance to Cowpea aphid-borne mosaic virus in species and hybrids of Passiflora: advances for the control of the passion fruit woodiness disease in Brazil

The potyvirus-induced passion fruit woodiness disease (PWD) is considered the most important limiting factor for passion fruit production in several countries. In Brazil, PWD is caused by the Cowpea aphid-borne mosaic virus (CABMV), and to date there are no reports on the existence of P. edulis genotypes resistant to this virus. Thus, resistance gene introgression from wild Passiflora species for a commercial species, via interspecific hybridization, is one of the strategies adopted in order to control the disease. The current study’s goals were to: confirm CABMV occurrence under field conditions; assess the resistance to CABMV in 178 Passiflora genotypes constituted by interspecific hybrids and their parents (P. edulis and P. setacea), as well as to estimate genetic parameters for the area under the disease progress curve (AUDPC), in order to obtain cultivars of sour passion fruit resistant to CABMV in future. The experimental design was set according to unbalanced randomized blocks with two repetitions. Data referring to the AUDPC were analyzed by means of the mixed models methodology (REMI/BLUP). CABMV infections were confirmed in sour passion fruit plants and in interspecific hybrids by observing foliar mosaic symptoms and by PTA-ELISA with specific antiserum against CABMV. There was a difference on the intensity of symptoms induced by CABMV for the 178 Passiflora genotypes assessed under natural occurrence conditions. The higher AUDPC values were obtained for 41 hybrids and for all P. edulis genotypes. In turn, lower values were estimated for 115 hybrid genotypes and for all P. setacea individuals. Of the 31 genotypes assessed by PTA-ELISA, 28 were considered resistant, out of those three P. setacea genotypes and 25 hybrids. Estimated AUDPC heritability values (0.99) and accuracy (0.99) enable inferring that resistance to CABMV within the assessed population was highly inheritable, allowing high selective efficiency. Resistant hybrid plants will be able to be selected and recombined with P. edulis genotypes and, again, assessed in order to corroborate the resistance to the virus, providing means of following up with the breeding genetic program on CABMV resistance.

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.

Erigeron bonariensis: hospedeira alternativa do Lettuce mosaic virus no Brasil

The genus Erigeron, Asteraceae family, comprises weed plants spread over Southern and Southeastern Brazil, and, frequently, is found among annual and perennial crop plants. Erigeron bonariensis L.plants showing symptoms of mosaic, similar to those caused by plant viruses, were collected in Sao Paulo State and submitted to electron microscopy, biological, serological and molecular analysis. Ultrathin sections of the original foliar tissues samples showed tubular and pinwheel inclusions dispersed in the cytoplasm of infected cells. Following transmission by mechanical inoculation, only Chenopodium amaranticolor, C. quinoa, Nicotiana benthamiana and N. clevelandii were infected. The ELISA results were negative with antisera against Turnip mosaic virus (TuMV) and Potato virus Y (PVY) and positive for Lettuce mosaic virus (LMV) antiserum. With specific primers to LMV, 280 bp fragments were amplified and sequenced, confirming the virus identity as LMV. The occurrence of LMV in E. bonariensis, which belongs to the lettuce (Lactuca sativa) family, is significant since it may also act as LMV reservoir for lettuce field crops. This is the first report in Brazil of a virus infecting Erigeron sp. which has also been reported as a natural host of Bidens mottle virus and Tomato spotted wilt virus in the United States.

Caracterização do Tomato chlorotic spot virus isolado de jiló no Vale do Paraíba, Estado de São Paulo

Tospoviruses are responsible for important losses in most crops, mainly Solanaceae. Gilo (Solanum gilo) plants showing mosaic, blistering, stunting and 100% production losses were collected for analysis from Sao Jose dos Campos in the State of Sao Paulo. Biological, electron microscopy, serological and molecular tests were carried out in order to characterize the virus isolate. The mechanical inoculation on Amaranthaceae, Solanaceae and Chenopodiaceae plants showed typical tospovirus-induced symptoms. Pleomorphic particles from 80 to 110 nm were observed in negatively stained preparations and in vesicles of the endoplasmic reticulum of infected cells. Tomato chlorotic spot virus (TCSV) was identified by DAS-ELISA. DNA fragments were amplified by RT-PCR, with specific primers designed to the nucleocapsid gene (N) of the main Tospovirus species, sequenced and compared with others in the GenBank. The nucleotide and amino acid deduced sequences homology was 99 and 95%, respectively, with TCSV. Comparison with other Tospovirus species presented values between 74 and 81%. These results confirmed the identity of this virus isolate as TCSV, the main tospovirus species in Sao Paulo that also damages other Solanaceous crops. Varieties of gilo have been inoculated showing susceptibility to TCSV, Tomato spotted wilt virus (TSWV) and Groundnut ringspot virus (GRSV).