First Report of Citrus leprosis virus on Citrus in Santa Cruz, Bolivia.

The citrus crop is rapidly expanding in the Province of Santa Cruz de la Sierra, Bolívia. Citrus, mostly planted by small growers, currently comprises approximately 15,000 ha. Sweet oranges (Citrus sinensis) and mandarins (C. reticulate) are the main citrus-types grown primarily for internal consumption. Recently, there has been an increase in incidence of leprosis-like symptoms (round to elliptic lesions on the leaves, chlorotic to necrotic lesions in young twigs, and depressed small lesions on the fruits). These symptoms were associated with infestations by the tenuipalpid mite Brevipalpus sp. To verify if Citrus leprosis virus was the causal agent of the observed symptoms, leaf and fruit samples (mostly from Valencia sweet orange) were collected from commercial groves in El Torno, 32 km south of Santa Cruz, and Yapacani and Colónia San Juan, 130 km northwest of Santa Cruz. Small fragments of these samples were placed immediately in a mixture of glutaraldehyde and paraformaldehyde in cacodylate buffer and later processed with transmission electron microscopy at ESALQ, Piracicaba, SP, Brazil. Some of the leaf samples were dried at 35°C and used for reverse transcription-polymerase chain reaction (RT-PCR) with primers that specifically amplify portions of the genome of Citrus leprosis virus, cytoplasmic type (CiLV-C) (1) at Centro APTA Citros, Cordeirópolis, SP, Brazil. Brevipalpus sp. mites were also collected and kept in 90% ethanol for further identification at the University of Florida, Gainesville and ESALQ. In the samples from the three surveyed areas, transmission electron microscopy confirmed the presence of short bacilliform particles within endoplasmic reticulum cisternae and electron dense viroplasms in the cytoplasm, typical of infection by CiLV-C (2). CiLV-C specific primers amplified DNA fragments of expected sizes in RT-PCR from dried leaf samples that came from these three localities. Direct sequencing of at least three amplicons of each sample confirmed the identity of the virus. The consensus sequence of the putative movement protein gene in samples from Yapacani and Colónia San Juan (GenBank Accessions Nos. AY960216 and AY960215, respectively) were identical and exhibited 99% nucleotide and 98% amino acid homology with the Brazilian isolate sequence available at GenBank (Accession No. AY289190). The consensus sequence of the putative replicase gene found in the sample from El Torno (GenBank Accession No. AY960214) exhibited 96 and 93% nucleotide and amino acid homology, respectively with the Brazilian isolate (GenBank Accession No. AY289191). Sampled mites were identified as B. phoenicis (Geijskes), the known vector of CiLV-C (2). The symptomatology, particle morphology and cytopathology, detection by molecular methods and the association with infestation by B. phoenicis, together indicate that the foliar, stem, and fruit lesions in sweet orange observed in the Santa Cruz region were caused by CiLV-C. To our knowledge, this is the first report of this virus in Bolivia. 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.