Antonio Carlos de Ávila

The identification of the vector species of iris yellow spot tospovirus occurring on onion in Brazil.

In Brazil, tospoviruses have been reported in several horticultural and ornamental plants. In the northeast region of Brazil, a tospovirus has emerged as a devastating virus on onion cultures. Based on serology and the sequence of nucleocapsid (N) protein gene, this pathogen was identified as a strain of iris yellow spot tospovirus (IYSV) (1). This virus was first identified on iris and leek in The Netherlands and later on onion in Israel. For an effective integrated management of tospoviruses in Brazil, identification of IYSV vector is essential. Three thrips species, Thrips tabaci, Frankliniella schultzei, and F. occidentalis, that are major vegetable and floral crop pests in the Federal District, Brazil, were tested for their ability to transmit the virus by leaf disk assay (2). All thrips, up to 8 h old, were given an acquisition access period of 48 h at 25°C on IYSV-infected Nicotiana benthamiana plants in Tashiro-cages. Thrips were then reared on uninfected Datura stramonium detached leaves until the adult stage. These adults were transferred individually to microcentrifuge tubes containing an N. benthamiana leaf disk and were incubated for 48 h for virus inoculation. The leaf disks were then incubated 4 more days to allow development of the virus infection, and the presence of virus was evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) with polyclonal antibodies against N protein of IYSV. Adult thrips were also used for direct inoculation to N. benthamiana plants, three thrips per plant. By the leaf disk assay, 45.8% (22 out of 48) of T. tabaci transmitted the virus, but F. schultzei (n = 48) and F. occidentalis (n = 32) did not transmit it. All plants (4 out of 4) directly inoculated by T. tabaci showed symptoms and infection by Dot-ELISA, while no plants inoculated with F. schultzei (n = 5) and F. occidentalis (n = 3) were positive, either by symptom observation or by Dot-ELISA. Only T. tabaci showed potential for a high capacity to transmit the IYSV onion isolate. In the field, considering the host preference of thrips, T. tabaci was considered the most important vector species of IYSV on onion. References: (1) L. Pozzer et al. Plant Dis. (In press.) (2) I. Wijkamp and D. Peters. Phytopathology 83:986, 1993.

Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virusfree garlic was evaluated.

First Report of Natural Occurrence of Zucchini Lethal Chlorosis Tospovirus on Cucumber and Chrysanthemum Stem Necrosis Tospovirus on Tomato in Brazil

During a field survey in 1994, five cucumber (Cucumis sativus) cv. Hokushin plants showing symptom of yellowing, mottling, and vein banding on the leaves were collected from a commercial field of the Federal District. By electron microscopy, quasi-spherical particles with double membrane, typical tospovirus-like particles were found in the infected leaf material. All samples strongly reacted with antibody of zucchini lethal chlorosis tospovirus (ZLCV), but not with antibodies of other to-spoviruses reported in Brazil (1): tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), groundnut ringspot virus (GRSV), chrysanthemum stem necrosis virus (CSNV), or iris yellow spot virusonion isolate (IYSV-BR). The virus was identified as ZLCV, which was first isolated in 1994 from zucchini (Cucurbita pepo) in São Paulo State, Brazil. Tomato (Lycopersicon esculentum) plants showing stem necrosis and necrotic spots and rings on the leaves were collected in Viçosa, Minas Gerais State. By electron microscopy, molecular studies, and enzyme-linked immunosorbent assay with antibodies of the six tospoviruses occurring in Brazil, the virus was identified as CSNV. This virus was first reported in 1995 on a Chrysanthemum sp. in São Paulo State and recently reported in the Netherlands from Dendranthema indicum. This is the first report of the natural occurrence of ZLCV and CSNV on cucumber and tomato, respectively. Reference: (1) A. C. de Ávila et al. 1998. Pages 32-34 in: Int. Symp. on Tospoviruses and Thrips in Floral and Vegetable Crops, 4th.

The N protein of Tomato spotted wilt virus (TSWV) is associated with the induction of programmed cell death (PCD) in Capsicum chinense plants, a hypersensitive host to TSWV infection.

In sweet pepper, the Tsw gene, originally described in Capsicum chinense, has been widely used as an efficient gene for inducing a hypersensitivity response (HR) derived Tomato spotted wilt virus (TSWV) resistance. Since previously reported studies suggested that the TSWV-S RNA mutation(s) are associated with the breakdown of Tsw mediated TSWV resistance in peppers, the TSWV genes N (structural nucleocapsid protein) and NS(S) (non-structural silencing suppressor protein) were cloned into a Potato virus X (PVX)-based expression vector, and inoculated into the TSWV-resistant C. chinense genotype, PI 159236, to identify the Tsw-HR viral elicitor. Typical HR-like chlorotic and necrotic lesions followed by leaf abscission were observed only in C. chinense plants inoculated with the PVX-N construct. Cytopathological analyses of these plants identified fragmented genomic DNA, indicative of programmed cell death (PCD), in mesophyll cell nuclei surrounding PVX-N-induced necrotic lesions. The other constructs induced only PVX-like symptoms without HR-like lesions and there were no microscopic signs of PCD. The mechanism of TSWV N-gene HR induction is apparently species specific as the N gene of a related tospovirus, Tomato chlorotic spot virus, was not a HR elicitor and did not cause PCD in infected cells.

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.

Estudo da interação de um begomovírus isolado de tomateiro com a mosca branca

A begomovirus named GO-ANPL (taxonomically related to the Tomato rugose mosaic virus, ToRMV), obtained from tomato (Lycopesricon esculentum) plants in Anapolis, state of Goias, was used to study virus/vector (Bemisia tabaci biotype B) interaction. The acquisition access period (AAP), the inoculation access period (IAP), and the latent period (LP) were determined by transferring five whiteflies per tomato seedling cv. Santa Clara. The minimum AAP was 15 min resulting in 6% infected plants, which reached 65% as the length of AAP increased to 24 h. At a minimum IAP of 30 min, 18% of plants were infected; this increased to 67% after an IAP of 24 h. The end of the LP in the vector occurred 16 h after virus acquisition. To detect the GO-ANPL in the vector by PCR, more than 2,500 specimens were tested. The virus was found in adults under different AAPs from the 1st to the 4th instar grown on infected plants. No virus was found in the eggs that were laid on infected plants by aviruliferous females. The GO-ANPL isolate was transmitted to the progeny of viruliferous females, since the virus was detected in eggs, all instars and adults. However, no virus transmission was observed from these adults. A high frequency of viral detection was observed in newly emerged adults from immature forms reared on virus-infected plants. These adults infected 33% of tomato plants in virus transmission assays. The results indicate that the interaction between begomovirus and B. tabaci biotipo B starts at early stages of insect development. This is important information for epidemiological studies of begomovirus in this country.

Ocorrência de viroses em tomate e pimentão na região serrana do estado do Espírito Santo

The highland area of the State of Espirito Santo is an important area for vegetable production comprising the counties of Vargem Alta, Venda Nova do Imigrante and Domingos Martins. A survey for viruses occurrence in the tomato and the sweetpepper crops was done in October, 2003. One hundred and thirty five leaf samples from tomato and sweetpepper showing virus-like symptoms were analyzed by serology and indicator hosts. Epidemics of Pepper Yellow mosaic virus, was observed in tomato and susceptible sweetpepper hybrids. Groundnut ring spot virus the only tospovirus species detected, and Cucumber mosaic virus was sporadically occurred. Potato virus Y, tobamoviruses and other tospoviruses were not detected in any of the analyzed leaf samples. Although high populations of the whitefly Bemisia argentifolii were present in tomato and sweetpepper no begomovirus-like symptoms were observed.

Distribuição de geminivírus nas culturas do tomate e pimentão em doze municípios do Submédio do Vale São Francisco

In 1996 and 1997, whitefly-transmitted geminivirus symptoms were observed in tomato (Lycopersicon esculentum) and sweet pepper (Capsicum annuum) plants in the Lower basin of San Francisco Valley, located in the states of Pernambuco and Bahia, Northeastern Brazil. One thousand three hundred and sixty-eight leaf samples of tomato and 194 pepper leaf samples showing similar symptoms to those caused by geminivirus were randomly collected from October 1996 to December 1998 in 104 and 16 fields, respectively, from 12 counties of that region and two neighboring counties. The incidence of symptomatic plants was estimated from 5 to 100% in tomato and 10 to 20% in sweet pepper fields. For geminivirus detection, dot or squash-blots were hybridized with heterologous probes. For tomato, the probe consisted of full-length DNA-A components of Bean golden mosaic virus (BGMV) from Brazil and Bean golden yellow mosaic virus (BGYMV) from Guatemala, while for sweet pepper it consisted of a fragment of the DNA-A component of an isolate from tomato found in the Federal District. Out of 1,562 collected samples, 908 (58.1%) tested positive for geminivirus, 823 (60.2%) on tomato and 85 (43.8%) on sweet pepper. The presence of infected plants was detected in all 120 fields with an incidence varying from 20% to 100%, indicating a broad dissemination of geminivirus in these crops in the Lower Basin of San Francisco Valley.

Evaluation of watermelon genotypes for resistance to Papaya ringspot virus, type watermelon

The aim of this study was to assess the resistance of nine watermelon genotypes against three PRSV-W isolates originated from three Brazilian States (Sao Paulo, Goias and Pernambuco). The experiment was carried out at Embrapa Hortalicas, Brasilia, Brazil, in April 2004. Nine watermelon genotypes were appraised, in a randomizated block design with four replications. Each plot was comprised of one 5 kg pot and five watermelon plants per pot. Ten to 13 days after sowing, inoculation was carried out with three PRSV-W isolates. Twenty-seven and 37 days after sowing, virus symptoms were evaluated. Virus presence or absence in the inoculated plants was confirmed by Das-Elisa. The results comprised variance analysis, heritability estimation, correlations among the characters, and genotype comparisons. Based on the different behavior of the genotypes in relation to each PRSV-W isolate, it is concluded that different isolates should be used in watermelon breeding programs. The high heritability values for most of the characters indicated that the characteristic in study is under the control of a few loci and, therefore, the possibility of selection of resistant watermelon accesses is high. The evaluated genotypes showed higher virus tolerance compared to the most planted cultivar in the country (Crimson Sweet), as it can be verified by the average values. The results suggest that the selected watermelon accesses are a good source of resistance to new watermelon cultivars tolerant to PRSV-W.

Ocorrência de vírus em batata em sete estados do Brasil

Viruses are responsible for the quick degeneration of potato seed-tubers. In the tropics, where aphid vectors are constantly present and the structure of virus populations is dynamic, the disease pressure is enormous. Therefore, the knowledge of such dynamics is definitely an extremely valuable tool towards the sustainability of the national potato production. In this study, we report a broad survey of virus occurrence in potato, in Brazil. In addition, we studied the distribution of the Potato virus Y (PVY) strains associated with mosaic symptoms on potatoes. In 2005 and 2006, we visited potato fields in seven Brazilian States. We collected leaves of symptomatic plants (1,256 samples) and also at random (360 samples). In addition, in several fields a visual assessment was carried out to estimate mosaic and leafroll incidence. From the 1,256 samples with symptoms, 840 tested serologically were positive to PVY (66.9%), 128 to PLRV (10.2%), 79 to PVS (6.3%), and none to PVX. Serology using DAS-ELISA and also biological and RT-PCR tests revealed an almost exclusive occurrence of the PVY necrotic strain, predominantly the necrotic subgroup PVYNTN. The analysis of a sub-sample representing all surveyed Counties indicated that the necrotic strain was universally present. Cultivars Asterix, Atlantic, Agata, Achat, Baronesa, Baraka, Bintje, Caesar, Cupido, Marijke, Monalisa, Panda, and Vivaldi, although displaying different susceptibility levels, were all infected by PVYNTN. The analysis of leaves collected at random showed similar results. PLRV was identified in four States (Minas Gerais, Bahia, Parana, and Santa Catarina), while PVS was present in the State of Sao Paulo as well. PVX was found in only one sample collected at random in Serra do Salitre, State of Minas Gerais. The contrast between visual evaluation and the results of the detection test by ELISA strongly indicated the presence of a relevant rate of PVY latent infection on cultivar Asterix.