D. F. Quito-Avila,
M. A. Ibarra,
R. A. Alvarez,
M. F. Ratti, and
E. L. Peralta, Centro de Investigaciones Biotecnológicas del Ecuador (CIBE), Escuela Superior Politécnica del Litoral (ESPOL), Campus Gustavo Galindo Km 30.5 vía Perimetral, apartado 09-01-5863, Guayaquil-Ecuador; and
R. R. Martin, USDA-ARS, Horticultural Crops Research Unit, Corvallis, OR, 97331
During the past two decades, several viruses have been identified from Rubus spp. in wild and commercial plantings around the world (2). In Ecuador, approximately 14 tons of blackberries are produced each year from an estimated area of 5,500 ha. In 2012, a preliminary survey was conducted to determine the presence of RNA viruses in Rubus glaucus, the most prevalent blackberry in Ecuador. Fifteen plants showing leaf mottling and severe mosaic were leaf-sampled from each of five different fields in Azuay Province. A total of 12 pooled samples of 20 g were obtained from the collected symptomatic tissue and used for dsRNA extraction using a cellulose-based protocol for detection of RNA viruses in plants (3). Three dsRNA segments of approximately 5 kbp, 2 kbp, and 900 bp were observed from all 12 dsRNA preparations. The dsRNA was heat-denatured and used as template for the generation of cDNA library using the universal random primer 5′-GCCGGAGCTCTGCAGAATTCNNNNNN-3′, for reverse transcription (RT), and the anchor primer 5′-GCCGGAGCTCTGCAGAATTC-3′for PCR as described (1). The PCR products were cloned using a StrataClone Kit (Agilent, CA) and sequenced (Macrogen, Korea). Sequence analysis revealed the presence of Raspberry bushy dwarf virus (RBDV), a pollen-borne Idaeovirus naturally found in several Rubus spp. worldwide. Approximately 120 RBDV sequences obtained from the Ecuadorean isolate were assembled into two contigs belonging to RNA1 and RNA2. Both sequences were re-confirmed by RT-PCR using specific primers. Partial sequences were assigned GenBank Accessions KC315894, KC315893, and KC315892 for the replicase, MP and CP, respectively. Furthermore, BLAST searches showed that the nucleotide sequence corresponding to the replicase was 95% similar to an isolate from the resistance breaking R15 strain (S51557.1), whereas the MP and CP nucleotide sequences were up to 98% similar to a Slovenian isolate (EU796088.1). Primers designed to amplify a 427-bp portion of the CP were used to detect RBDV from four blackberry plantings in two distant production areas: Ambato in Tungurahua Province and Paute in Azuay Province. Leaf mottling and severe mosaic was observed in 90% of blackberry fields in those two locations. Leaf samples (n = 90) were randomly collected from both symptomatic and asymptomatic plants in each location. In Ambato, RBDV was detected in 50% and 40% of symptomatic and asymptomatic plants, respectively. In Paute, RBDV was present in 70% of symptomatic plants and 29% of asymptomatic plants. The presence of RBDV in asymptomatic plants suggests the virus might not be the sole causal agent of the disorder. Further studies are needed to determine the role of RBDV in the observed symptoms, since virus complexes responsible for increased severity of symptoms have been commonly reported in Rubus spp. (4). R. glaucus is native to the tropical highlands (from Ecuador to Mexico) and differs from blackberries commercially grown in the United States and Europe. Therefore, RBDV-induced symptoms reported in blackberry grown in the United States and Europe may not be extrapolated to the Andes berry. To the best of our knowledge, this is the first report of RBDV from blackberry in Ecuador.
References: (1) P. Froussard. Nucleic Acids Res. 20:2900, 1992. (2) R. R. Martin et al. Plant Dis. 97:168, 2013. (3). T. J. Morris and J. A. Dodds. Phytopathology 69:854. 1979. (4) D. F. Quito-Avila et al. J. Virol. Methods 179:38, 2012.