Identification and Characterization of Citrus Chlorotic Spot Virus, a New Dichorhavirus Associated with Citrus Leprosis-Like Symptoms.

Local chlorotic spots resembling early lesions characteristic of citrus leprosis (CL) were observed in leaves of two sweet orange (Citrus sinensis L.) trees in Teresina, State of Piauí, Brazil, in early 2017. However, despite the similarities, these spots were generally larger than those of a typical CL and showed rare or no necrosis symptoms. In symptomatic tissues, transmission electron microscopy revealed the presence of viroplasms in the nuclei of the infected parenchymal cells and rod-shaped particles with an average size of approximately 40 × 100 nm, resembling those typically observed during infection by dichorhaviruses. A bipartite genome of the putative novel virus, tentatively named citrus chlorotic spot virus (CiCSV) (RNA1 = 6,518 nucleotides [nt] and RNA2 = 5,987 nt), revealed the highest nucleotide sequence identity values with the dichorhaviruses coffee ringspot virus strain Lavras (73.8%), citrus leprosis virus N strain Ibi1 (58.6%), and orchid fleck virus strain So (56.9%). In addition to citrus, CiCSV was also found in local chlorotic lesions on leaves of the ornamental plant beach hibiscus (Talipariti tiliaceum (L.) Fryxell). Morphological characterization of mites recovered from the infected plants revealed at least two different types of Brevipalpus. One of them corresponds to Brevipalpus yothersi. The other is slightly different from B. yothersi mites but comprises traits that possibly place it as another species. A mix of the two mite types collected on beach hibiscus successfully transmitted CiCSV to arabidopsis plants but additional work is required to verify whether both types of flat mite may act as viral vectors. The current study reveals a newly described dichorhavirus associated with a citrus disease in the northeastern region of Brazil.

Brugmansia suaveolens mottle virus, a novel potyvirus causing leaf mottling of Brugmansia suaveolens in Brazil.

A potyvirus was isolated from Brugmansia suaveolens showing leaf mottling and tentatively named Brugmansia suaveolens mottle virus (BsMoV). The virus (isolate Bs-Campinas) could infect some solanaceous plants and two Chenopodium species, and was transmitted by aphids. Symptomatic leaves contained flexuous particles and cylindrical inclusions. RT-PCR amplification using potyvirus universal primers produced a DNA fragment of 1851 nt (3' terminal genomic region), which shared 71% nucleotide identity with Pepper mottle virus, the best-matched potyvirus sequence. Since this identity value is below the threshold currently used to discriminate Potyvirus species, Brugmansia suaveolens mottle virus most likely represents a new Potyvirus species.

Natural Infection of Swinglea glutinosa by the Citrus leprosis virus Cytoplasmic Type (CiLV-C) in Colombia.

Swinglea glutinosa (Blanco) Merr., a perennial plant in the family Rutaceae, is originally from southeast Asia but which is now grown worldwide. In Colombia, it is used as an ornamental and principally as a living fence around rural properties and farms in several regions of the country. Citrus leprosis virus cytoplasmic type (CiLV-C) was recently detected in orange groves of the Colombian Piedmont eastern plains, an area known as the Llanos Orientales (2). Because of the potential for country-wide infection of citrus, some measures are being taken to avoid CiLV-C spread to other regions of Colombia. Further surveys made from June to December 2005 to evaluate the extent of the spread of CiLV-C in the Llanos Orientales revealed some plants in S. glutinosa hedges surrounding citrus orchards exhibiting chlorotic spots and ringspots of varied size on the leaves, similar to those caused by CiLV-C on sweet oranges leaves. These plants were found near citrus orchards in the municipalities of Guamal and in some urban areas of Villavicencio City in the Meta Department. The possibility that these symptoms were caused by CiLV-C was investigated soon after sample collection by the same procedures as described previously for sweet orange (2). In the leaf lesions of S. glutinosa, typical bacilliform particles and dense cytoplasmic viroplasm were found with electron microscopy. Total RNA extracted from symptomatic leaves was subjected to reverse transcription-PCR (RT) using primers (Fwd. 5'GATACGGGACGCATAACA-3'/Rev. 5'-TTCTGGCTCAACATCTGG-3') that specifically amplify a region within the CiLV-C putative methyltransferase gene and this yielded a single fragment of the expected 402 bp (3). Analysis of the consensus sequence derived from 20 RT-PCR products (GenBank Accession No. EU689106) showed 96% nucleotide and 92% amino acid sequence identity to the sequence of a Brazilian CiLV-C isolate (GenBank Accession Nos. DQ352194.1 and YP_654565.1), respectively. Recently, published work described mite transmission of CiLV-C to some nonrutaceous plants (1), but to our knowledge, this is the first report of a nonCitrus rutaceous plant naturally infected by CiLV-C. Mites found in citrus orchards and previously identified as Brevipalpus phoenicis (Geijskes) (2), which are likely the most important vector of CiLV-C in citrus in Colombia, were observed feeding on healthy and symptomatic S. glutinosa, indicating that S. glutinosa is a host for B. phoenicis. Because the use of S. glutinosa as a living fence or hedge is a common practice in Colombia, CiLV-C-infected S. glutinosa plants may play a role in the epidemiology of leprosis in commercial citrus by serving as an inoculum source for this lethal virus. References: (1) M. Bastianel et al. Summa Phytopathol. 32:211, 2006. (2) G. A. León et al. Plant Dis. 90:682, 2006. (3) E. C. Locali et al. Plant Dis. 87:1317, 2003.

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$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.