Identification of Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus infecting Lactuca sativa with symptoms of lettuce big-vein disease in Argentina

Lettuce big-vein disease (BVD) affects all major lettuce-producing areas of the world. The causal agent is Mirafiori lettuce big-vein virus (MLBVV), an ophiovirus transmitted by the soil-borne fungus Olpidium brassicae (Lot et al., 2002). MLBVV has been detected in many different areas of the world but never in Argentina. La Plata has about 700 ha of lettuce with a production of about 13,000 t, and with about 70% of the total production from Buenos Aires Province. BVD has been detected in different areas in the north and west of the La Plata horticultural green belt. Many of the plants with BVD symptoms had leaf distortions of moderate severity, which affected their commercial value (Fig. 1). Over the winters of 2007, 2008 and 2009, forty samples of lettuce with BVD symptoms, representing most of the commercial varieties, were taken from fields near La Plata. Samples tested positive by RT-PCR with universal primers for the genus Ophiovirus (Vaira et al., 2003) and/or by DAS-ELISA using a polyclonal antiserum against the viral coat protein of MLBVV (kindly provided by Dr Y. Kawazu, National Institute of Vegetable and Tea Science, Japan). 

The identity of MLBVV was verified by cloning and sequencing amplicons (GenBank Accession Nos. FJ555204-05), which shared 97% identity with both Italian (AY204674.1) and Brazilian (DQ854813.1) MLBVV isolates.Specific primers, Cps1 (5´-CTCATGACAAAAGAAGAGAAGC) and Cpas1 (5´-CACATCAAATATGAAGTTGTGCTC) were designed from MLBVV-RNA 3, to allow specific MLBVV detection in RNA extracts from collected samples.The primers were tested using samples with symptoms that were positive by RT-PCR and DAS-ELISA. An amplicon of the expected 450 bp was amplified from all infected samples.Sequence analysis (GU295451) demonstrated high identity (98%) with sequences deposited in the public databases.A block sampling method was employed to collect random field samples and estimate the incidence of the disease. These were scored for symptoms and tested for infection by RT-PCR (primers Cps1/Cpas1).In the three lots tested, the incidence of the disease reached 60% of plants inoculated.Additionally, soil transmission experiments were conducted using healthy lettuce seedlings planted into contaminated soil or by watering with rinse water from the roots of diseased field plants. After five weeks in a greenhouse up to 70% of the 15 seedlings inoculated with five field isolates showed big-vein symptoms and tested positive by DAS-ELISA and RT-PCR. Three of the plants used for transmission were also positive for Lettuce big-vein associated virus (LBVaV), detected by RT-PCR using specific primers (VP248 and VP249; Navarro et al., 2004).This is the first report confirming the presence of MLBVV and LBVaV infecting lettuce plants in Argentina.