Gilvan Pio-Ribeiro

Cowpea aphid-borne mosaic virus (CABMV) is widespread in passionfruit in Brazil and causes passionfruit woodiness disease

Summary.Leaf samples of yellow passionfruit (Passiflora edulis f. flavicarpa) displaying fruit woodiness symptoms were collected in seven Brazilian states and the Federal District. Viral infection was confirmed by host range and ELISA, and fourteen viral isolates were obtained. All isolates were capable of infecting several leguminous host species, although differences in symptom severity were noticeable. Woodiness symptoms were reproduced in yellow passionfruit, and mosaic symptoms were induced in common bean. All isolates infected cowpea, reported as a non-host of passion fruit woodiness virus (PWV). Indirect ELISA demonstrated that all isolates were serologically related to each other and also to cowpea aphid-borne mosaic virus (CABMV). The complete sequence of the capsid protein was determined for all isolates. Comparison of these sequences with those of other potyviruses indicated the highest identity with CABMV isolates (85 to 94%). Identity with PWV isolates ranged from 54 to 70%. Phylogenetic analysis grouped all of the Brazilian isolates in a monophyletic cluster with the CABMV isolates, clearly distinct from the PWV isolates. Furthermore, this analysis demonstrated that a group of previously characterized isolates from Brazil that had been designated as PWV should be reclassified as CABMV. Together, these results provide unequivocal evidence that, in Brazil, passionfruit woodiness disease is primarily caused by CABMV. The presence of PWV in Brazil has yet to be confirmed.

Stock indexing and Potato virus Y elimination from potato plants cultivated in vitro

Potato cultivars (Solanum tuberosum L.) have shown degeneration or run out caused by viruses after several cycles of propagation using seed tubers from commercial fields. This work reports the occurrence of single and mixed infections of four potato viruses in Paraiba-Brazil and presents a method for Potato virus Y (PVY) elimination, by using thermo-and chemotherapies. Plants of potato cv. Baraka were tested by direct antigen coating ELISA. Antisera against PVY, Potato virus X (PVX), Potato virus S (PVS), and Potato leafroll virus (PLRV) were used. Materials with positive reaction to PVY were treated for virus elimination. Single node cuttings (1.0 cm length) were excised and inoculated in Murashige & Skoog (MS) medium, supplemented with 1.0 mg L-1 of kinetin, 0.001 mg L-1 of naphthalene acetic acid (NAA) and 0.1 mg L-1 of gibberellic acid (GA3). The thermotherapy at approximately 37oC, during 30 and 40 days, resulted in 20.0 and 37.5% PVY elimination, respectively. Chemotherapy was undertaken with Ribavirin (RBV), 5-Azacytidine (AZA), and 3-Deazauridine (DZD). The RBV showed the highest rate of virus eradication, with 55.5% virus-free plants. Simultaneous thermo and chemotherapy had higher efficiency for the elimination of PVY, reaching rates of healthy plants of 83.3% with RBV, 70.0% with AZA, and 50.0% with DZD.

Análise Filogenética de Potyvírus Causando Endurecimento dos Frutos do Maracujazeiro no Nordeste do Brasil

Leaf samples of yellow passionfruit plants (Passiflora edulis f. flavicarpa) showing woodiness symptoms in the fruit were collected in fields located in the states of Pernambuco, Paraiba and Sergipe. Virus infection was confirmed by serology and inoculation of indicator plants. The six viral isolates obtained from passionfruit were capable of infecting several plant species, although a difference in the intensity of symptoms induced by each isolate was observed in some hosts. Indirect ELISA showed that the passionfruit isolates were serologically related to each other, and also to the potyvirus Cowpea aphid-borne mosaic virus (CABMV). The amino acid sequence of the capsid protein was determined for the six isolates. Sequence comparisons with other potyviruses indicated a maximum degree of identity with CABMV (86 to 94%). Sequence identity with isolates of Passionfruit woodiness virus (PWV) ranged from 68 to 76%. Phylogenetic analysis based on the amino acid sequences confirmed that the Brazilian passionfruit isolates are closely related to CABMV, and only distantly related to PWV. Together, these results indicate that the potyvirus isolates obtained from passionfruit comprise a strain of CABMV.

High genetic variability and recombination in a begomovirus population infecting the ubiquitous weed Cleome affinis in northeastern Brazil

Diseases caused by begomoviruses are a serious constraint to crop production in many tropical and subtropical areas of the world, including Brazil. Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that are often associated with weed plants, which may act as natural reservoirs of viruses that cause epidemics in crop plants. Cleome affinis (family Capparaceae) is an annual weed that is frequently associated with leguminous crops in Brazil. Samples of C. affinis were collected in four states in the northeast of Brazil. Analysis of 14 full-length DNA-A components revealed that only one begomovirus was present, with 91-96% identity to cleome leaf crumple virus (ClLCrV). In a phylogenetic tree, ClLCrV forms a basal group relative to all other Brazilian begomoviruses. Evidence of multiple recombination events was detected among the ClLCrV isolates, which also display a high degree of genetic variability. Despite ClLCrV being the only begomovirus found, its phylogenetic placement, high genetic variability and recombinant nature suggest that C. affinis may act as a source of novel viruses for crop plants. Alternatively, ClLCrV could be a genetically isolated begomovirus. Further studies on the biological properties of ClLCrV should help to clarify the role of C. affinis in the epidemiological scenario of Brazilian begomoviruses.

Sequences of the coat protein gene from brazilian isolates of Papaya ringspot virus

Papaya ringspot virus (PRSV) is the causal agent of the main papaya (Carica papaya) disease in the world. Brazil is currently the worlds main papaya grower, responsible for about 40% of the worldwide production. Resistance to PRSV on transgenic plants expressing the PRSV coat protein (cp) gene was shown to be dependent on the sequence homology between the cp transgene expressed in the plant genome and the cp gene from the incoming virus, in an isolate-specific fashion. Therefore, knowledge of the degree of homology among the cp genes from distinct PRSV isolates which are present in a given area is important to guide the development of transgenic papaya for the control of PRSV in that area. The objective of the present study was to assess the degree of homology among the PRSV cp genes of several Brazilian isolates of this virus. Papaya and PRSV are present in many different ecosystems within Brazil. Twelve PRSV isolates, collected in eight different states from four different geographic regions, were used in this study. The sequences of the cp gene from these isolates were compared among themselves and to the gene used to generate transgenic papaya for Brazil. An average degree of homology of 97.3% at the nucleotide sequence was found among the Brazilian isolates. When compared to 27 isolates from outside Brazil in a homology tree, the Brazilian isolates were clustered with Australian, Hawaiian, and Central and North American isolates, with an average degree of homology of 90.7% among them.

ARRANJO ESPACIAL DO VIRA-CABEÇA DO FUMO EM ARAPIRACA, ESTADO DE ALAGOAS*

The spotted wilt of tobacco (Nicotiana tabacum), caused by Groundnut ringspot virus (GRSV), genus Tospovirus, has been observed with high severity in certain areas of the tobacco producing region of Alagoas. The spatial pattern of the disease was analysed in two areas (A and B), formed by four parcels each, located in the county of Arapiraca. The parcels were evaluated weekly by spatial mapping of healthy and spotted wilt tobacco plants, as well as by the disease incidence, represented by the number of plants with symptoms in relation to the total evaluated plants. Through the analyses of ordinary runs and spatial autocorrelation, a random pattern diseased plants was predominantly observed, although aggregation was also detected in a few cases. Although the possible effect of infected seedlings in the spatial pattern was not discarded, the most probable hypothesis is that the infections mainly occurred due to the primary inoculum transmitted by viruliferous thrips entering the parcels from external reservoirs.

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.

Detecção de allexivírus em primórdios foliares de alho via RT-PCR

Cloves crop is a routinely practice often used in procedures for detecting allexiviruses in garlic bulbs to obtainment foliar tissue to be analyzed by serological and/or molecular tests. The availability of the plants under greenhouse implies is expenses with the maintenance and requires abaut 30 days. In areas free of these viruses, there is also the risk of their introduction and dissemination. This study presents an adjustment of protocol aiming a fast detection of allexiviruses, using primordial leaves. Cloves of consumption-garlic, from Rio Grande do Sul, Brazil, and imported from Argentina were dissected for obtaining primordial leaves and total RNA extraction, using 0,1g of tissue of each sample. RT-PCR reactions were performed with a pair of primers able to amplify a 500 bp fragment, corresponding to the internal region of the coat protein gene from several species of Allexivirus genus. A band in the expected height (500 pb) was visualized in agarose gels and further confirmed using Southern Blot test and by sequencing as Garlic virus C (GarV-C, AY170322.1). Total RNA obtained from foliar primordia of cloves and its use in RT-PCR analysis is an economic, fast and secure methodology for allexivirus detection in garlic bulbs.

Complete genome sequence of melon yellowing-associated virus from melon plants with the severe yellowing disease in Brazil

Here, we describe the complete genome sequence of melon yellowing-associated virus (MYaV), found in melon plants with severe yellowing disease, determined by high-throughput and Sanger sequencing. MYaV has an RNA genome of 9073 nucleotides plus a poly(A) tail. At least six open reading frames were predicted, with a typical carlavirus genomic organisation. Phylogenetic analysis of the complete genome sequence and the amino acid sequences of the RNA-dependent RNA polymerase confirmed that MYaV belongs to the genus Carlavirus, with the highest genome-wide nucleotide sequence identity of 59.8% to sweet potato yellow mottle virus.

Desempenho agronômico de videiras com e sem sintomas de viroses, e comparação molecular de isolados virais

The objective of this work was to evaluate the effect of viroses in symptomatic and asymptomatic grapevines on the agronomic variables related to the plant vigor and the enological quality of grapes, and to compare viral isolates obtained from these two conditions. Two experiments were performed with four cultivars. All plants were indexed by real time, reverse transcription‑polymerase chain reaction (RT‑PCR) for the probable occurrence of the following viruses: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine leafroll‑associated virus (GLRaV‑1 to ‑4, GLRaV‑4 strain 5), Grapevine rupestris stem pitting‑associated virus (GRSPaV), and Grapevine fleck virus (GFkV). The evaluated variables were: number of buds burst and not burst, number of branches with or without bunches, total number of buds, number of bunches, fresh bunch weight, total berry weight, rachis weight, number of berries per bunch, average berry weight, total soluble solids, total titratable acidity, pH, pruned branch weight, or rootstock and scion trunk diameters. The negative effects were more pronounced on the plants with virus symptoms; however, it was observed that plants without symptoms were also often infected. The molecular analysis of GRSPaV, GVA, and GLRaV‑2, isolates from symptomatic and asymptomatic plants, resulted in a high percentage of nucleotide identities between homologous isolates.