ABSTRACT Zinnia sp. and hairy beggartick (Bidens pilosa) plants exhibiting symptoms of possible virus infection were found in the municipality of Santa Bárbara d’Oeste, São Paulo State, Brazil. Flexuous filamentous particles and cytoplasmatic inclusions typical of potyvirus infection were observed by transmission electron microscopy, respectively, in leaf extracts and cells of symptomatic leaves. Infection of both plants with bidens mottle virus (BiMoV) was confirmed by RT-PCR using potyvirus universal primers, followed by nucleotide sequencing of the amplicons. The nearly complete genome sequence of the Brazilian isolate, named BiMoV-BR, is 9700 nucleotides long and shares 95.6 % identity with the corresponding nucleotide sequence of a BiMoV isolate from the United States. BiMoV-BR was mechanically transmitted and caused systemic infection on plants of Zinnia sp., hairy beggarstick, sunflower (Helianthus annuus), and lettuce (Lactuca sativa). Myzus persicae transmitted the virus to Zinnia sp. plants with efficacy of 8 % and 42 %, using one and ten aphids per plant, respectively. This is the first detection of BiMoV in Brazil. Further studies are necessary to evaluate the distribution of this potyvirus in the country.

RESUMO A alface (Lactuca sativa L.) pertencente à família Asteraceae e é uma das hortaliças mais consumidas no Brasil. O Lettuce mosaic virus (LMV) é um dos vírus mais importantes da cultura e, atualmente, a principal forma de controle é a utilização de genótipos portadores do gene de tolerância mol1 e mo12. Um isolado de LMV proveniente de Botucatu (SP), denominado LMV-Bot, foi identificado quanto as suas características biológicas e moleculares. Ao ser inoculado nas variedades de alface diferenciadoras de patótipos de LMV em alface (‘Ithaca - gene Mo’, ‘Malika - mo11’, ‘Vanguard 75 – Mo e mo12’ e ‘Salinas 88 - mo12’), o LMV-Bot induziu sintomas somente em ‘Salinas 88’. A porção N’ terminal que codifica a proteína capsidial foi amplificada, sequenciada e seu posicionamento filogenético revelou similaridade com dois isolados brasileiros denominados Br6 e A435 e também com um coletado na França (Fr4). Porém, o LMV-Bot não se enquadrou no sub-grupo Most e Common. Os resultados sugerem que apesar de raros, isolados atípicos de LMV são encontrados na natureza infectando alface, e podem contornar o gene de resistência mol2.

ABSTRACT Lettuce (Lactuca sativa L.) belongs to the Asteraceae family and is one of the most consumed vegetables in Brazil. Lettuce mosaic virus (LMV) an important virus for this crop and, currently, its main control form is based on the utilization of genotypes carrying the tolerance gene mol1 and mol2. A LMV isolate from Botucatu (SP), named LMV-Bot, was identified considering its biological and molecular characteristics. When inoculated in distinctive lettuce varieties that classifies LMV pathotypes (‘Ithaca - Mo gene’, ‘Malika – mol1’ ‘Vanguard 75 - Mo and mol2’ and ‘Salinas 88 - mo12’), LMV-Bot induced symptoms only in ‘Salinas 88’. The N’ terminal coding region for the capsid protein was amplified and sequenced, and its phylogenetic position revealed similarity to two Brazilian isolates named BR6 and A435 and to another one from France (FR4). However, LMV-Bot did not fit in the sub-group Most and Common. Results suggest that, although rare, atypical isolates of LMV are found in nature infecting lettuce and can break the resistance gene mol2.

Resumo: O objetivo deste trabalho foi avaliar genótipos de alface quanto à reação ao Lettuce mosaic virus (LMV; Most-type, isolado AF-199) e variações no fator eucariótico de tradução eIF4E. Todos os genótipos inoculados foram suscetíveis ao LMV, que foi detectado por RT-PCR com primers específicos. Porém, os acessos 169501, 169501C, 172918A e 162499, apresentaram sintomas tardios e somente nas folhas inoculadas. O sequenciamento da porção codificadora para eIF4E mostrou que estes genótipos apresentam padrão eIF4E0 (mol0), típico para suscetibilidade ao LMV, o que indica que o fenótipo encontrado não está correlacionado com as variações de nucleotídeos neste fator de tradução.

Abstract: The objective of this work was to evaluate lettuce genotypes for their reaction to Lettuce mosaic virus (LMV; Most-type, isolate AF-199) and variations of the eukaryotic translation initiation factor eIF4E. All inoculated genotypes were susceptible to LMV, which was detected by RT-PCR using specific primer pairs. However, the accessions 169501, 169501C, 172918A, and 162499 showed late development of symptoms that appeared only on the inoculated leaves. Sequencing of the coding region of eIF4E showed that these genotypes have an eIF4E0 (mol 0 ) standard typical for their susceptibility to LMV, indicating that the phenotype found is not correlated to nucleotide variations in this translation factor.

Resumo: O objetivo deste trabalho foi avaliar o efeito da aplicação de fungicidas na concentração de Tomato severe rugose virus (ToSRV, Begomovirus) no híbrido de tomateiro 'Mariana' coinfectado por ToSRV e Tomato chlorosis virus (ToCV, Crinivirus) e a progressão da concentração viral por qPCR, bem como quantificar a produção e a qualidade de frutos. O experimento I consistiu na aplicação dos fungicidas após a semeadura (pré-tratamento): piraclostrobina+metiram (P+M) (a 3 g L-1) + boscalida (B) (a 0,3 g L-1), seguida de pulverizações quinzenais com P+M (4 g L-1); no experimento II, não houve aplicação na semeadura (controle), apenas 4 g L-1 de P+M quinzenalmente. As transmissões de ToSRV e ToCV foram realizadas com uso de moscas-brancas (Bemisia tabaci, biótipo B), aos 15, 30, 45, 60 e 70 dias após o transplante (DAT). Houve aumento da produção, melhor qualidade dos frutos e redução da concentração de ToSRV nas plantas, quando os vírus foram transmitidos tardiamente e o pré-tratamento foi realizado. O pré-tratamento na bandeja, na semeadura, com P+M (3 g L-1) e B (0,3 g L-1), seguido de pulverizações quinzenais com P+M (4 g L-1), aumenta a produção e a qualidade dos frutos, em tomateiro híbrido 'Mariana' coinfectado aos 45, 60 e 75 DAT por ToSRV e ToCV, e há redução da concentração de ToSRV.

Abstract: The objective of this work was to evaluate the effect of fungicide application on the concentration of Tomato severe rugose virus (ToSRV, Begomovirus) in the 'Mariana' hybrid tomato coinfected with ToSRV and Tomato chlorosis virus (ToCV, Crinivirus) and the progression of viral concentration by qPCR, as well as to quantify fruit yield and quality. Experiment I consisted in the application of fungicides after sowing (pretreatment): pyraclostrobin+metiram (P+M) (at 3 g L-1) + boscalid (B) (at 0.3 g L-1), followed by biweekly sprayings with P+M (4 g L-1); in experiment II, there was no application at sowing (control treatment), only 4 g L-1 P+M biweekly. ToSRV and ToCV transmissions were performed using sweetpotato whiteflies (Bemisia tabaci, biotype B), at 15, 30, 45, 60, and 70 days after transplanting (DAT). There was an increase in yield, better fruit quality, and a reduction in the ToSRV concentration in the plants, when the viruses were transmitted late and pretreatment was performed. Tray pretreatment in sowing with P+M (3 g L-1) and B (0.3 g L-1), followed by biweekly sprayings with P+M (4 g L-1), increases fruit yield and quality in the 'Mariana' hybrid tomato coinfected at 45, 60, and 75 DAT by ToSRV and ToCV, and there is a reduction in the concentration of ToSRV.

ABSTRACT Identification of Thysanoptera is based mainly on external morphology examination that can be time-consuming and difficult for non taxonomic experts. In this work, we propose a rapid and efficient molecular method to identify Frankliniella schultzei, an important and widespread pest thrips vector of tospoviruses in South America countries. Species-specific primers designed in the mitochondrial cytochrome oxidase I gene (mtCOI) were optimized for detection by conventional PCR and real-time PCR. The primers were tested on immature and adult thrips collected from crops and weeds found in São Paulo State. All samples collected were identified as F. schultzei, indicating the high prevalence of this species as vector of tospoviruses in Brazilian fields.

A mosca branca Bemisia tabaci (Hemiptera: Aleyrodidae) é considerada uma das pragas agrícolas mais ameaçadoras em todo o mundo, pelo seu efeito direto na sucção de seiva das plantas e principalmente pelo seu efeito indireto de transmitir vírus, em especial os begomovírus. No Brasil, a espécie de begomovírus predominante em solanáceas é o Tomato severe rugose vírus - ToSRV. Uma possibilidade de manejo da doença é a utilização de cultivares resistentes ou tolerantes tanto ao vírus, como ao inseto vetor. Foram avaliados neste trabalho a dispersão primária e secundária do ToSRV por Bemisia tabaci, espécie MEAM1, também conhecida como biótipo B, em cultivares tolerantes ao ToSV Capsicum baccatum (IAC-1357) C. annuum (IAC-1566) e C. frutescens (IAC-1544). O genótipo (IAC-1544) apresentou menor número de plantas infectadas pelo ToSRV no ensaio de dispersão primária e nenhuma eficiência na transmissão do isolado na dispersão secundária. Estes resultados colaboram para o melhoramento de pimentão e pimentas visando a resistência ao ToSRV e seu inseto vetor.

The whitefly Bemisia tabaci (Hemiptera : Aleyrodidae) is considered one of the most threatening agricultural pests in the world due to its direct effect on the suction of sap from plants and especially due to its indirect effect in transmitting viruses, particularly the begomovirus. In Brazil, the predominant species of begomovirus within Solanaceae is Tomato severe rugose virus - ToSRV. A possibility of management of this disease is the use of cultivars that are resistant or tolerant to both the virus and the insect vector. This study evaluated the primary and secondary dispersal of ToSRV by B. tabaci, MEAM1 species, also known as biotype B, usin ToSRV-tolerant species Capsicum baccatum (IAC-1357), C. annuum (IAC -1566) and C. frutescens (IAC-1544). The genotype IAC -1544 showed a smaller number of plants infected by ToSRV during the primary dispersal assay and no efficiency in the isolate transmission in the secondary dispersal. These results contribute to capsicum and pepper breeding forresistance to ToSRV and its insect vector.

Garlic plants are naturally infected by a mixture of viruses, including allexiviruses. Symptomatic garlic plants with mosaic and distorted leaves from garlic producing regions in Brazil were analyzed for the presence of Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C), Garlic virus D (GarV-D) and Garlic mite-borne filamentous virus (GarMbFV), five allexivirus species previously reported in the country. Fifty-nine virus isolates from five distinct allexivirus species were identified and the complete coat protein region of each genome was sequenced. Mixed infections were very frequent and corresponded to 43% of the positive samples. The nucleotide identity of the coat protein ranged between 75% and 98% for GarV-A isolates, 83% and 90% for GarV-B, 69% and 98% for GarV-C, 87% and 97% for GarV-D, and 72% and 91% for GarMbFV.

As plantas daninhas podem atuar como importantes reservatórios de vírus. Solanum americanum (Maria Pretinha) é uma planta daninha comum no Brasil e amostras com mosaico foram coletadas em áreas produtoras de pimentão, para verificar a presença de vírus. Uma amostra apresentou infecção mista com Cucumber mosaic virus (CMV) e Potato virus Y (PVY) e uma amostra apresentou infecção simples com PVY. As duas espécies virais foram transmitidas por extrato vegetal e ocasionaram mosaico em tomate (Solanum lycopersicum cv. Santa Clara), pimentão (Capsicum annuum cv. Magda), Nicotiana benthamiana e N. tabaccum TNN, lesões locais em Chenopodium quinoa, C. murale e C. amaranticolor. As sequências de proteína capsidial para o CMV e PVY encontradas em S. americanum estão filogeneticamente mais relacionadas com isolados de tomate. Conclui-se que S. americanum pode atuar como reservatório de diferentes vírus, durante e entre as épocas de cultivo de pimentão.

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.

Tomato severe rugose virus (ToSRV) is the predominant species of begomovirus in São Paulo State, Brazil, and infects primarily tomato and pepper plants. There is no information about genetic resistance of pepper to this virus, so in this work the reaction of 29 genotypes of Capsicum spp. was evaluated by inoculation of two ToSRV isolates: ToSRV-Sk (isolated from a tomato plant) and ToSRV-PJU (isolated from a pepper plant). For both isolates, two C. annuun genotypes (Catarino Cascabel - México and Silver) showed no symptoms 30 days after inoculation (d.a.i). In a second experiment, these two genotypes were evaluated for 150 d.a.i and, again, no symptoms could be observed. However, the virus was detected by RCA-PCR, indicating that both genotypes are susceptible, but less affected by ToSRV infection. Catarino Cascabel - México and Silver can be indicated for use in breeding programs for resistance of pepper to ToSRV.

Entre os problemas fitossanitários da cultura da alface estão as doenças causadas por vírus. Três vírus causam sintomas de mosaico praticamente indistinguíveis: o Lettuce mosaic virus (LMV, Potyvirus), o Lettuce mottle virus (LeMoV, Sequivirus) e o Bidens mosaic virus (BiMV, Potyvirus). Através de RT-PCR utilizando-se oligonucleotídeos específicos para cada um destes vírus, amostras de alface e plantas invasoras, preferencialmente com sintoma de mosaico, foram coletadas em campos de produção de alface das regiões de Mogi das Cruzes, Campinas e Bauru no Estado de São Paulo e analisadas para a presença dos vírus. Verificou-se que o LeMoV foi o vírus encontrado com maior freqüência, seguido do LMV. A ocorrência de BiMV em alface foi extremamente baixa e restrita às regiões de Campinas e Bauru, onde também foi verificado em plantas invasoras como Bidens pilosa e Galinsoga parviflora. Esta ultima é hospedeira dos três vírus.

Among the phytosanitary problems affecting lettuce culture are the diseases caused by viruses. Three viruses cause almost indistinguishable mosaic symptoms: Lettuce mosaic virus (LMV, Potyvirus), Lettuce mottle virus (LeMoV, Sequivirus) and Bidens mosaic virus (BiMV, Potyvirus). By RT-PCR using specific primers for each virus, lettuce and weed samples, preferably with mosaic symptoms, and collected from lettuce fields in the regions of Mogi das Cruzes, Campinas and Bauru, São Paulo State, Brazil, were analyzed for the presence of these viruses. LeMoV was the most frequently found virus, followed by LMV. The occurrence of BiMV in lettuce was extremely low and restricted to the regions of Campinas and Bauru, where it was also found in weeds such as Bidens pilosa and Galinsoga parviflora. The latter plant is the host of these three viruses.

O Potato virus Y (PVY) e Pepper yellow mosaic virus (PepYMV) são as únicas espécies de potyvirus encontradas em pimenta e pimentão no Brasil. A região codificadora para a proteína capsidial de isolados de PepYMV e PVY coletados em pimentão, foi avaliada quanto à variabilidade e presença de motivos específicos aos potyvirus. A identidade da seqüência de aminoácidos na CP entre os isolados de PepYMV foi de 93% a 100%, enquanto que para os de PVY 94% a 98%. Entre os vírus esta variou de 73% a 79%. Foi observada variabilidade nas regiões conservadas da CP. Todos os isolados de PepYMV seqüenciados não apresentaram o motivo DAG na CP, relacionada a transmissão dos vírus por afídeos, enquanto que para as seqüências obtidas de PVY foi observada. Demais domínios como MVWCIENG, ENTERH, QMKAAA e PYMPRYG foram verificadas em ambas espécies.

Potato virus Y (PVY) and Pepper yellow mosaic virus (PepYMV) are the only potyvirus species found in pepper and sweetpepper in Brazil. The region encoding the coat protein of PVY and PepYMV isolates collected in sweetpepper was evaluated for the presence of specific motifs of potyviruses. The identity of the amino acid sequence in CP among PepYMV isolates was 93% to 100%, while for PVY was 94% to 98%. Among the viruses that ranged from 73% to 79%. It was observed variability in the conserved regions of the CP. All PepYMV isolates sequenced did not show the DAG motif in the CP, related to virus transmission by aphids, while for sequences of PVY this motif was observed. Other motifs, such MVWCIENG, ENTERH, QMKAAA and PYMPRYG were found in both species.

Bemisia tabaci (Genn.) é considerada uma das mais importantes pragas em cultivos de hortaliças e ornamentais em todo o mundo. Baseado na análise da seqüência mitocondrial (citocromo oxidase I - mtCOI) foi proposto recentemente que B. tabaci deva ser considerado um complexo críptico de espécies, contendo 11 grupos e 24 espécies. Dois destes grupos: Middle East-Asia Minor e Mediterranean englobam os biótipos B e Q, respectivamente. Avaliou-se a sequência mtCOI de espécimes de B. tabaci coletados em regiões do estado de São Paulo, Brasil. Por PCR-RFLP utilizando-se a enzima Taq I, pôde-se observar somente o padrão típico de clivagem para o biótipo B. Comparando-se com sequências consenso, todas as moscas brancas foram classificadas no grupo Middle East-Asia Minor e puderam ser separadas em quatro haplótipos, indicando prevalência do biótipo B em áreas de pimentão (Capsicum annuum L.), tomate (Solanum lycopersicum L.), cucurbitáceas e berinjela (Solanum melongena L.) do Estado de São Paulo.

Bemisia tabaci (Genn.) is one of the most important pests in cultivated areas of vegetables and ornamental crops around the world. Based on the mitochondrial cytochrome oxidize I (mtCOI) sequence, there is evidence that B. tabaci should be considered a cryptic species complex of 11 groups containing 24 species. Two of the groups, Middle East-Asia Minor 1 and Mediterranean include biotypes B and Q, respectively. In this study we evaluated the mtCOI sequence of B. tabaci populations collected in sites of the state of São Paulo, Brazil. Using PCR-RFLP with Taq I, a typical biotype B profile was obtained for all specimens. Based on the comparison with mtCOI reference sequences we found four haplotypes all belonging to the Middle East-Asia Minor 1. They occurred in the hosts pepper (Capsicum annuum L.), tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.) and cucurbitaceae plants.

O Bidens mosaic virus (BiMV) é uma espécie tentativa do gênero Potyvirus, que infecta alface (Lactuca sativa). Na ausência de métodos eficientes para diagnose deste vírus, o objetivo do trabalho foi a síntese de oligonucleotídeos específicos e sua otimização em testes de RT-PCR em uma só etapa, partindo-se de extrações de RNA total. Os oligonucleotídeos 8851sens (5'AGG CAG TTC GCA CGG CAT AC 3´) e 9211ant (5´ CTT CAT CTG GAT GTG TGC TTC 3´) permitem a eficiente detecção do vírus e possibilitaram a descoberta de uma nova hospedeira do vírus, a planta Galinsoga parviflora, comumente encontrada em canteiros de produção comercial de alface.

Bidens mosaic virus (BiMV) is a tentative species of the genus Potyvirus and infects lettuce (Lactuca sativa). In the absence of efficient methods for the diagnosis of the virus, the objective of this work was to develop specifics primers for a one-step RT-PCR test using total RNA. The primer pairs 8851sens (5'AGG CAG TTC GCA CGG CAT AC 3´) and 9211ant (5´ CTT CAT CTG GAT GTG TGC TTC 3´) were considered very efficient for the detection of BiMV and the virus was found naturally infecting Galinsoga parviflora, a new host of BiMV, commonly found in commercial lettuce crops.

Vírus do gênero Begomovirus são transmitidos por mosca-branca Bemisia tabaci G., e constituem um dos problemas fitossanitários sérios em diversas culturas. Plantas de pimentão coletadas em oito regiões do Estado de São Paulo, foram submetidas a extração de DNA total e PCR com primers universais e degenerados para begomovírus, que amplificam parte da região codificadora para a proteína capsicial. Os dados indicam a presença de begomovírus em pimentão nas cinco regiões coletadas. Análise das seqüências do DNA viral e análise filogenética revelaram identidade com dois begomovírus nativo da América. Tomato severe rugose virus - ToSRV (AY029750) e com Tomato yellow vein streak virus (ToYVSV, AY829113), espécies descritas infectando tomateiro no Brasil. A presença de begomovírus em pimentão foi verificada nas regiões de Alvinlândia, Ubirajara, Botucatu, Elias-Fausto, Paulínia, Mogi Guaçu, Paranapanema e Pirajú.

Virus to the genus Begomovirus transmitted by whiteflies Bemisia tabaci G., and considered one of the most important fitossanitary problems for several cultures. Pepper plants were collected in eight the regions of São Paulo State, the extraction of total DNA and PCR were submitted with universal and degenerate primers was used to amplify partial sequences of the begomovírus coat protein gene. The data indicated the presence of begomovírus in five collected regions. DNA viral sequence analysis and phylogenetic analysis revealed identity with two native begomovírus of America. Tomato severe rugose virus - ToSRV (AY029750) and with Tomato yellow vein streak virus (ToYVSV, AY829113), described species infecting tomateiro in Brazil. The begomovírus presence in pepper plant was verified in the areas of Alvinlândia, Ubirajara, Botucatu, Elias-Fausto, Paulínia, Mogi Guaçu, Paranapanema and Pirajú.