Renato B. Salaroli

The significance of changes in Mytella falcata (Orbigny, 1842) gill filaments chronically exposed to polluted environments

The impact of pollutants in an organism can be observed by changes in functional complexity at different levels. Bivalve gills are suitable for histopathological analysis because of their structure and function. This study aimed at examining the morphology of Mytella falcata gill filaments from three sites in the Santos estuary (São Paulo, Brazil) with different levels of environmental degradation to identify possible changes in gill structure and discuss the significance of these alterations. For this purpose, histological, histochemical and ultrastructural techniques were used. The filaments of animals from site A (less impacted site) were intact, while in sites B and C, pathological changes were observed, such as: detachment of the epithelium in the intermediate zone, morphological changes of this epithelium, inflammatory process, increase in the number of mucous cells and cell turnover processes. These results suggest that the related changes are an attempt to prevent the entrance of pollutants through gill filaments into the entire organism and that cell turnover is the final way to compensate cell injury.

Occurrence of Citrus leprosis virus in Llanos Orientales, Colombia

In Colombia, citrus is cultivated in mostly small plantings that total 55,000 ha by approximately 25,000 farmers. Production includes 1,200 tons of fresh fruits and 60 tons of juice for domestic consumption, resulting in a net worth of US

Chlorotic spots on Clerodendrum, a disease caused by a nuclear type of Brevipalpus (Acari:Tenuipalpidae) transmitted virus

650,000 per year. Most of the production comes from areas located between the Cordillera Occidental and Cordillera Central mountain ranges (departments of Antioquia, Caldas, Quindio, and Risaralda) near coffee plantations. The departments of Meta and Casanare, located at the east plains (Llanos Orientales), include a zone parallel (4 to 5°N, 72 to 74°W) to the east mountain range and generate approximately 10% of the total Colombian citrus production. Suspected citrus leprosis symptoms on leaves and fruits of sweet oranges (Citrus sinensis (L.) Osb.) were first observed by plant pathologists for CORPOICA (Colombian National Agricultural Research Organization) in citrus orchards in Casanare in 2003, and later in 2004, in Meta. To confirm the visual identification, leaves and fruits from Valencia sweet orange exhibiting typical lesions of leprosis were collected from several locations in the departments of Casanare (Yopal, Aguazul) and Meta (Guamal, Villavicencio, and Cumaral). Samples were fixed in cacodylate-buffered paraformaldehyde/glutaraldehyde solution and subsequently processed for examination in thin sections using electron microscopy. Samples were processed and examined at the Citrus Research and Educational Center (CREC) of the University of Florida, Lake Alfred, and the Agricultural College (ESALQ) of the Universidade de São Paulo at Piracicaba, SP, Brazil. Some leaf samples collected in Meta were also dried and used for detection of Citrus leprosis virus, cytoplasmic type (CiLV-C) by reverse transcription-polymerase chain reaction (RT-PCR) at the Centro APTA Citros Sylvio Moreira at Cordeirópolis (CAPTACSM). The RT-PCR was performed with primers that specifically amplify a fragment of the viral genome that codes for the putative cell-to-cell movement protein (1). Locations at CREC and ESALQ each observed, using electron microscopy, cell changes characteristic of CiLV-C that include short bacilliform particles in the endoplasmic reticulum and dense, vacuolated, and irregularly shaped viroplasm in the cytoplasm (2) in samples from Casanare and Meta. RT-PCR amplified cDNA fragments of the expected size for samples collected in Meta and one of the amplicons was sequenced (GenBank Accession No. DQ272491). The sequence obtained was found to have 98% nucleotide sequence identity to the Brazilian CiLV-C isolate (GenBank Accession No. AY289190.1). Mites collected from affected plants from the department of Meta were identified at ESALQ as Brevipalpus phoenicis (Geijskes), a known principal vector of CiLV-C (2). These several lines of evidence confirmed that the symptoms observed in sweet oranges at Meta and Casanare are due to the infection by CiLV-C. To our knowledge, this is the first report of this virus in Colombia. References:(1) E. C. Locali et al. Plant Dis. 87:1317, 2003, (2) J. C. V. Rodrigues et al. Exp. Appl. Acarol. 30:161, 2003.

Experimental host range of Citrus leprosis virus C (CiLV-C)

Manchas cloroticas e necroticas foram observadas em folhas de varias plantas de coracao-sangrento (Clerodendrum x speciosum) cultivadas em parques e jardins em Piracicaba, SP, associadas a infestacao pelo acaro tenuipalpideo Brevipalpus phoenicis. Exames preliminares de seccoes de tecido das manchas cloroticas ao microscopio eletronico revelaram a ocorrencia de efeitos citopaticos caracteristicos dos induzidos pelos virus do tipo nuclear, transmitido por acaros Brevipalpus (VTB). Brevipalpus phoenicis coletados de C. x speciosum sintomatico e transferidos para plantas sadias de C. x speciosum reproduziram as lesoes. O acaro tambem transmitiu o patogeno para C. thomsonae, Gomphrena globosa, Hibiscus cannabinus, H. coccineus, H. schizopetalus, Salvia leucantha, Spathiphyllum wallasi e Tetragonia expansa, as quais exibiram manchas cloroticas e/ou necroticas. O virus tambem foi transmitido mecanicamente para Chenopodium amaranticolor, C. quinoa, G. globosa, H. cannabinus, H. coccineus e T. expansa, alem de C. x speciosum. Plantas de C. amaranticolor e C. quinoa mantidas a 28 - 30oC desenvolveram infeccao sistemica. Em todos os tecidos sintomaticos das plantas-teste inoculadas, examinados ao microscopio eletronico, foram encontrados efeitos citopaticos do tipo nuclear causado por VTB. O virus foi purificado a partir de folhas com infeccao sistemica de C. amaranticolor e C. quinoa. Injecoes de preparacoes purificadas em coelho geraram um anti-soro policlonal que reagiu especificamente com o antigeno homologo em teste de ELISA. As evidencias obtidas indicam que as manchas cloroticas do Clerodendrum estao associadas a um VTB do tipo nuclear, tentativamente denominado de virus da mancha clorotica do Clerodendrum (Clerodendrum chlorotic spot virus- ClCSV).

Brevipalpus-associated viruses in the central Amazon Basin

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 virus N: A New Dichorhavirus Causing Citrus Leprosis Disease

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.

Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C expressed in Escherichia coli: production and use in immunodiagnosis

Citrus leprosis (CL) is a viral disease endemic to the Western Hemisphere that produces local necrotic and chlorotic lesions on leaves, branches, and fruit and causes serious yield reduction in citrus orchards. Samples of sweet orange (Citrus × sinensis) trees showing CL symptoms were collected during a survey in noncommercial citrus areas in the southeast region of Brazil in 2013 to 2016. Transmission electron microscopy analyses of foliar lesions confirmed the presence of rod-like viral particles commonly associated with CL in the nucleus and cytoplasm of infected cells. However, every attempt to identify these particles by reverse-transcription polymerase chain reaction tests failed, even though all described primers for the detection of known CL-causing cileviruses and dichorhaviruses were used. Next-generation sequencing of total RNA extracts from three symptomatic samples revealed the genome of distinct, although highly related (>92% nucleotide sequence identity), viruses whose genetic organization is similar to that of dichorhaviruses. The genome sequence of these viruses showed <62% nucleotide sequence identity with those of orchid fleck virus and coffee ringspot virus. Globally, the deduced amino acid sequences of the open reading frames they encode share 32.7 to 63.8% identity with the proteins of the dichorhavirids. Mites collected from both the naturally infected citrus trees and those used for the transmission of one of the characterized isolates to Arabidopsis plants were anatomically recognized as Brevipalpus phoenicis sensu stricto. Molecular and biological features indicate that the identified viruses belong to a new species of CL-associated dichorhavirus, which we propose to call Citrus leprosis N dichorhavirus. Our results, while emphasizing the increasing diversity of viruses causing CL disease, lead to a reevaluation of the nomenclature of those viruses assigned to the genus Dichorhavirus. In this regard, a comprehensive discussion is presented.

Present status of citrus leprosis in Argentina and Paraguay

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.

Tomato spotted wilt virus infects spider lily plants in Australia

Citrus leprosis (CL) was first described in South America in the 1920s. 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 Boqueron, Concepcion, San Pedro, Cordillera, Alto Parana, Itapua) and Argentina (Provincias de Misiones, Corrientes, Entre Rios). 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.

Fine structure of Mytella falcata (Bivalvia) gill filaments.

Tomato spotted wilt virus (TSWV) was identified in green red-rimmed ringspots on spider lily (Hymenocallis spp.) leaves in Brisbane, Australia. Tospovirus-like particles were seen in thin sections of those lesions. RT-PCR using tospovirus-specific primers amplified a DNA fragment whose sequence matched TSWV S RNA. The virus caused symptoms in Nicotiana benthamiana following mechanical transmission and was confirmed as TSWV by RT-PCR.