Citrus leprosis (CL) is a serious threat to the citrus industry, especially for sweet oranges. For a long time, Citrus spp. were considered the only susceptible hosts. However, other plant species were also found either experimentally or naturally to be susceptible to Citrus leprosis virus C (CiLV-C). To assess the experimental host range of CiLV-C, a large number of plant species were inoculated with Brevipalpus phoenicis, viruliferous to CiLV-C, under experimental conditions. Out of the 140 tested species (43 families), 59 species (24 families) developed localized chlorotic and/or necrotic lesions upon inoculation of leaves with viruliferous mites, and 40 species (18 families) of them yielded positive results for CiLV-C detection in at least one of the following assays: ELISA, RT-PCR, transmission electron microscopy and immunfluorescence. For those that developed lesions and yielded negative results in CiLV-C detection assays, the results may be attributed to the small number of lesions and their necrotic state with very little viral material. The fact that a considerable number of plant species are susceptible to the virus after mite inoculation brings up implications for the epidemiology, quarantine and evolution of the citrus leprosis pathosystem.

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.

Foi feito um levantamento no estado do Amazonas, na região da capital Manaus, na base de exploração de petróleo de Urucu (700 km a sudoeste de Manaus) e em Presidente Figueiredo (100 km ao norte de Manaus) à procura de vírus transmitidos por ácaros Brevipalpus (VTrB) em diferentes espécies de plantas. Foram coletadas 33 espécies de plantas caracterizadas por lesões localizadas em folhas (manchas cloróticas, manchas anelares, manchas verdes em folhas senescentes) e pela presença de ácaros Brevipalpus (os quais foram coletados para identificação e avaliação de sua diversidade) em plantas cultivadas, ornamentais e silvestres. Fragmentos das lesões foram fixados no local e processados para microscopia eletrônica ultraestrutural e examinados em Piracicaba, SP. Foram constatados treze casos positivos de detecção de efeitos citopáticos do tipo citoplasmático ou nuclear de VTrB nas plantas coletadas em Manaus. Três deles parecem representar casos anteriormente descritos de VTrB em Hibiscus rosa sinensis, H. syriacus e Clerodendrum x speciosum. Presumíveis VTrB do tipo nuclear foram constatados em mancha clorótica em Piper callosum (Piperaceae), mancha clorótica anelar em Monstera deliciosa (Araceae), mancha clorótica em Ruellia chartacea (Acanthaceae), manchas cloróticas em Bidens sp. (Asteraceae), mancha verde em Allamanda chatartica (Apocynaceae), mancha clorótica em Gardenia sp. (Rubiaceae), mancha verde em Mussaenda erythrophylla (Rubiacea) e do tipo citoplasmático em mancha clorótica em Clerodendrum speciosissimum (Lamiaceae) e mancha anular em Arundina graminifolia (Orchidaceae). Todas estas plantas achavam-se naturalmente infestadas por Brevipalpus phoenicis.

A survey of Brevipalpus-transmitted plant viruses (BTrV) was carried out in the cities of Manaus and Presidente Figueiredo, and in the oil exploratory base of Urucu, Amazonas State, Central Amazon Basin, Brazil. The main characteristics considered during the survey were the occurrence of localized symptoms (chlorotic or brown spots, ringspots, green spots in senescent leaves) similar to those previously described for BTrV and infestation by Brevipalpus mites. For the evaluation of the infection by putative BTrV, small fragments of the leaf lesions were fixed in loco and later processed for ultrastructural electron microscopy at Piracicaba, SP. Thirteen plant hosts of presumed BTrV were found. Three of them probably represent previously described BTrV infecting Hibiscus rosa sinensis, H. syriacus and Clerodendrum x speciosum. For the first time we report cases of infection by putative BTrV of nuclear type in chlorotic spot in Piper callosum (Piperaceae), chlorotic ringspot in Monstera deliciosa (Araceae), chlorotic spot in Ruellia chartacea (Acanthaceae), chlorotic spot in Bidens sp. (Asteraceae), green spot in Allamanda chatartica (Apocynaceae), chlorotic spot in Gardenia sp. (Rubiaceae), chlorotic ringspot and green spot in Mussaenda erythrophylla. (Rubiaceae) and of cytoplasmic type in Clerodendrum speciosissimum (Lamiaceaae) and ringspots in Arundina graminifolia (Orchidaceae). All these plants were infested by Brevipalpu. phoenicis.