Citrus leprosis (CL) was first described in South America in the 1920's. It is considered similar to a disease first observed back to 1860 in Florida. It is a destructive disease characterized by localized lesions on the leaves, fruits and stems, which may lead to the death of the affected plant if left untreated. Around 1940, CL was demonstrated to be transmitted by Brevipalpus mites in Argentina. Because little information exists on the status of CL pathosystem in Argentina and Paraguay, a survey was made in several citrus growing areas of these countries from 2009 to 2011, to evaluate its presence and relevance as well as the identification of the virus and the mite vector. CL was found in most of the sweet orange and/or mandarin orchards in Paraguay (Departamentos de Boquerón, Concepción, San Pedro, Cordillera, Alto Paraná, Itapúa) and Argentina (Provincias de Misiones, Corrientes, Entre Ríos). Incidence was usually low. The causal virus was identified as Citrus leprosis virus C (CiLV-C) by RT-PCR, electron microscopy and immunofluorescence. In all the visited regions in Paraguay and the region of Montecarlo, Argentina, the mites collected in plants infected by CiLV-C were identified as B. phoenicis.

This work reports the in vitro expression of Citrus leprosis virus C (CiLV-C) putative coat protein (p29) and the production of a polyclonal antibody to be used as a tool for serological diagnosis of citrus leprosis. The ORF2/RNA1, corresponding to p29, was cloned in pET28a and transformed into Escherichia coli cells (BL21). Expression of p29 was induced in vitro and the protein was purified and used for immunization of rabbits to produce the polyclonal antibody. The anti p29 serum was shown to be highly specific to CiLV-C detection by immunological methods (Western blot, PTA-ELISA, tissue blot and in situ immunolocalization), without cross reaction with healthy citrus plants or other cytoplasmic and nuclear viruses transmitted by Brevipalpus mites. These results demonstrate that the antibody against CiLV-C p29 protein is highly specific for CiLV-C detection. In situ immunogold labeling assays on thin sections of sweet orange leaf cells infected by CiLV-C demonstrated that short, bacilliform particles present within cisternae of the endoplasmic reticulum were specifically labeled, confirming their viral nature. The dense cytoplasmic viroplasm was also heavily labeled indicating that it represents a site of p29 accumulation.

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

A análise da variabilidade genética de geminivírus infetando tomateiros (Lycopersicon esculentum) na região Sudeste do Brasil foi realizada por meio do seqüenciamento direto de fragmentos de PCR amplificados com oligonucleotídeos universais para o componente A de begomovírus, seguido de análise filogenética. Amostras de tomateiro e de algumas plantas daninhas associadas, apresentando sintomas típicos de infecção por vírus, foram coletadas em sete municípios nos estados de Minas Gerais, Espírito Santo e Rio de Janeiro. Após extração de DNA total e PCR, verificou-se que 137 amostras estavam infetadas por begomovírus, de um total de 369 amostras coletadas. A análise filogenética indicou existir uma grande diversidade genética de begomovírus em tomateiros nas três regiões amostradas, com a predominância de uma espécie viral (Tomato chlorotic mottle virus, TCMV) na Zona Metalúrgica de MG, enquanto no RJ e ES predomina uma outra espécie não completamente caracterizada. A análise filogenética indicou ainda a presença de outras quatro possíveis novas espécies de begomovírus em tomateiro. O alto grau de diversidade genética encontrado sugere uma transferência recente de begomovirus a partir de hospedeiros silvestres para o tomateiro. O relacionamento filogenético encontrado entre os begomovirus de tomateiro e isolados virais obtidos a partir de plantas daninhas favorece essa hipótese.

The genetic diversity of begomovirus isolates from tomato (Lycopersicon esculentum) fields in the Southeastern region of Brazil was analyzed by direct sequencing of PCR fragments amplified by using universal oligonucleotides for the begomovirus DNA-A, and subsequent computer-aided phylogenetic analysis. Samples of tomato plants and associated weeds showing typical symptoms of virus infection were collected at seven locations in the states of Minas Gerais, Espírito Santo and Rio de Janeiro. A total of 137 out of 369 samples were infected with a begomovirus based on PCR analysis. Phylogenetic analysis indicated a high degree of genetic diversity among begomoviruses infecting tomatoes in the sampled area. One species (Tomato chlorotic mottle virus, TCMV) occurs predominantly in Minas Gerais, whereas in Rio de Janeiro and Espírito Santo a distinct species, not yet fully characterized, predominates. Phylogenetic analysis further indicates the presence of an additional four possible new species. This high degree of genetic diversity suggests a recent transfer of indigenous begomovirus from wild hosts into tomatoes. The close phylogenetic relationship verified between begomovirus infecting tomato and associated weeds favors this hypothesis.