Em 2005 houve um relato da presença da leprose dos citros no Estado de Chiapas, ao sul do México, baseado em sintomatologia. Subsequentemente a doença foi constatada em pomares de laranjeiras doces no Estado de Tabasco, nas localidades de Huimanguillo e Cunduacan. Amostras de folhas com lesões lepróticas de laranjeiras Valencia ou Hamlin foram coletadas e posteriormente examinadas ao microscópio eletrônico e submetidas a ensaios de detecção molecular por RT-PCR usando primers específicos. Estudos ultraestruturais revelaram alterações citopatológicas típicas causadas pelo CiLV-C (partículas baciliformes curtas no lúmen do retículo endoplasmático e a presença de viroplasmas vacuolados e elétron-densos no citoplasma) e hipertrofia em algumas células do parênquima lacunoso. Ensaios de RT-PCR resultaram na amplificação, na maioria das amostras de folhas dessecadas, de fragmento de DNA de tamanho esperado. Ácaros Brevipalpus coletados nos pomares foram identificados como B. phoenicis. Ensaios de transmissão com este ácaro confirmaram seu papel como vetor do CiLV-C para laranja doce, mas não houve transmissão para limão, limão Volkameriana, citrange C35 e citrange Carrizo. Todos estes dados considerados em conjunto confirmam que CiLV-C está presente em Tabasco, México e que a leprose dos citros descrita em Chiapas é causada pelo CiLV-C.

Citrus leprosis was detected in sweet oranges in Chiapas, Mexico, in 2005 based on symptoms. The disease was soon after observed in sweet orange orchards at Huimanguillo and Cunduacan, state of Tabasco. Leaf samples of leprosis-affected Valencia or Hamlin sweet oranges were collected and subjected to ultrastructural examination and molecular detection of CiLV-C by RT-PCR. Cytopathic effects typical of CiLV-C infection as presence of short, baciliform particles in the endoplasmic reticulum cisternae and electron dense, vacuolated viroplasma in the cytoplasm, as well as hypertrophy of some spongy parenchyma cells were observed in the tissues of the leaf lesions. RT-PCR using specific primers for CiLV-C produced DNA fragments of the expected size. Brevipalpus mites were present in these orchards and identified as B. phoenicis, the known vector for CiLV-C. Transmission tests with these mites confirmed their role as leprosis vectors to sweet orange but not to lemon, Volkamerian lemon, C35 citrange and Carrizo citrange. These data confirm the presence of CiLV-C in Tabasco, Mexico and that the citrus leprosis found in Chiapas was caused by CiLV-C.

Coffee ringspot is a minor coffee disease caused by the nuclear type of Brevipalpus mite-transmitted virus, Coffee ringspot virus (CoRSV). Recently outbreaks of the disease in some growing regions of the state of Minas Gerais, Brazil, were registered with qualitative and quantitative yield losses. Coffea arabica was the only species registered as natural host. A survey was made on a germplasm collection of Coffea and related species kept at the Centro de Café "Alcides Carvalho", Instituto Agronômico, Campinas, state of São Paulo (SP), Brazil, to assess natural susceptibility of Coffee species, other than C. arabica and some interspecific hybrids of Coffea as well as other non-Coffea plant species to the Coffee ringspot virus (CoRSV). The following plants were found with ringspot symptoms on their leaves and/or fruits besides C. arabica L.: C. kapakata (IAC 4511), C. dewevrei cv. Excelsa, C. canephora cv. Robusta, hybrid derivative of the C. arabica × C. racemosa (IAC1195-5-6-2), C. arabica × C. dewerei (Piatã IAC 387), Híbrido de Timor CIFC 832/1 (derivative from a natural crossing between C. arabica × C. canephora) and C. racemosa. Also Psilanthus ebracteolatus, a species close to the genus Coffee was also found with ringspot lesions on their leaves. All these plants were also found infested by Brevipalpus mites identified as B. phoenicis. Infection of these plants by CoRSV was confirmed by the observation of characteristic cytopathic effects in the tissues of the lesion and by RT-PCR using a pair of primer specific for CoRSV. Only with C. racemosa RT-PCR failed to amplify the CoRSV genome. The susceptibility of P. ebracteolatus to CoRSV adds new dimension regarding its controversial taxonomic position.

Solano-violeta (Solanum violaefolium) é uma planta ornamental rasteira usada para cobrir solos de áreas sombreadas. Um vírus que induz manchas anelares nas folhas desta planta, tentativamente designado Solanum violaefolium ringspot virus - SvRSV, transmitido pelo ácaro Brevipalpus phoenicis (Acari: Tenuipalpidae) foi encontrado em Piracicaba, SP. Trata-se de um vírus baciliforme que se assemelha a outros vírus do tipo citoplasmático transmitidos por Brevipalpus sp. Este trabalho teve como objetivo relatar propriedades biológicas e estabelecer uma caracterização molecular parcial do SvRSV. O vírus pode ser transmitido mecanicamente a várias outras espécies botânicas, causando lesões localizadas. Entre as espécies avaliadas, Datura stramonium mostrou-se a melhor hospedeira experimental. Observou-se também a manifestação de sintomas nestas plantas após infestação das mesmas por B. obovatus previamente alimentado em lesões de SvRSV, confirmando esta outra espécie de ácaro como vetor do vírus. Suas propriedades físicas in vitro foram: temperatura de inativação 40-45 ºC; ponto final de diluição 10-3-10-4; longevidade in vitro 12 dias. Em secções ultrafinas, as partículas do SvRSV mostraram-se levemente mais delgadas e mais longas que as de outros vírus do mesmo grupo. A partir do dsRNA do SvRSV foi construída uma biblioteca de cDNA e foram identificadas duas possíveis regiões codificadoras das proteínas de movimento e replicase viral. Baseado nestas regiões foram desenhados "primers" para amplificação do RNA do SvRSV por RT-PCR. Sondas baseadas nas seqüências obtidas hibridizaram com ss- e dsRNA de D. stramonium infectadas pelo vírus. Ensaios preliminares de RT-PCR e hibridização não resultaram em reação com o vírus da leprose dos citros, tipo citoplasmático (CiLV-C).

Solanum violaefolium is an ornamental plant, with prostrate, trailing growth habit and is cultivated in shaded areas. A virus that causes ringspot symptoms on its leaves, tentatively named as Solanum violaefolium ringspot virus (SvRSV) and transmitted by Brevipalpus phoenicis (Acari: Tenuipalpidae) was found in Piracicaba city, São Paulo State. It is a bacilliform virus that resembles other cytoplasmic viruses transmitted by Brevipalpus sp. The objective of this work is to describe the biological properties and establish partial molecular characterization of the SvRSV. The SvRSV can be transmitted mechanically to several plant species causing local lesions. Among the tested species, Datura stramonium was proved to be the best experimental host. It was observed that S. violaefolium plants were infested by B. obovatus that also transmitted SvRSV in preliminary assays. Its in vitro physical properties were: thermal inactivation point: 40-45 ºC; dilution end point: 10-3-10-4 and in vitro longevity: 12 days. Ultrathin sections revealed that SvRSV particles are slightly thinner and longer than other cytoplasmic-type viruses transmitted by Brevipalpus sp. A cDNA library from dsRNA molecules extracted from SvRSV was constructed and two regions, which putatively code for the replicase and movement proteins were identified. Based on these sequences, primer pairs were designed for SvRSV´s RNA amplification by RT-PCR. Indeed, probes based on such sequences hybridized with ss- and dsRNA from infected D. stramonium plants. Preliminary molecular assays using primers and probes of SvRSV did not detect Citrus leprosis virus, cytoplasmic type (CiLV-C), another cytoplasmic type Brevipalpus-transmitted viruses.

In order to obtain a better understanding of what is citrus, 33 cDNA libraries were constructed from different citrus species and genera. Total RNA was extracted from fruits, leaves, flowers, bark, seeds and roots, and subjected or not to different biotic and abiotic stresses (pathogens and drought) and at several developmental stages. To identify putative promoter sequences, as well as molecular markers that could be useful for breeding programs, one shotgun library was prepared from sweet orange (Citrus sinensis var. Olimpia). In addition, EST libraries were also constructed for a citrus pathogen, the oomycete Phythophthora parasitica in either virulent or avirulent form. A total of 286,559 cDNA clones from citrus were sequenced from their 5’ end, generating 242,790 valid reads of citrus. A total of 9,504 sequences were produced in the shotgun library and the valid reads were assembled using CAP3. In this procedure, we obtained 1,131 contigs and 4,083 singletons. A total of 19,200 cDNA clones from P. parasitica were sequenced, resulting in 16,400 valid reads. The number of ESTs generated in this project is, to our knowledge, the largest citrus sequence database in the world.