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

A.E. BARCALA TABARROZZI; E.J. PEÑA; E. DAL BO; G. ROBLES LUNA; C.A. REYES; M.L. GARCIA

PLANT PATHOLOGY

WILEY-BLACKWELL PUBLISHING, INC

Lugar: Londres; Año: 2010 vol. 59 p. 1160 - 1161

0032-0862

Lettuce big-vein disease (BVD) affects all major lettuce-producing areasof the world. The causal agent is Mirafiori lettuce big-vein virus(MLBVV), an ophiovirus transmitted by the soil-borne fungus Olpidiumbrassicae (Lot et al., 2002). MLBVV has been detected in many differentareas of the world but never in Argentina. La Plata has about 700 ha oflettuce with a production of about 13 000 tonnes, and with about 70% ofthe total production from Buenos Aires Province. BVD has been detectedin different areas in the north and west of the La Plata horticultural greenbelt. Many of the plants with BVD symptoms had leaf distortions of moderateseverity, which affected their commercial value. Over the winters of2007, 2008 and 2009, forty samples of lettuce with BVD symptoms, representingmost of the commercial varieties, were taken from fields nearLa Plata. Samples tested positive by RT-PCR with universal primers forthe genus Ophiovirus (Vaira et al., 2003) and ⁄ or by DAS-ELISA using apolyclonal antiserum against the viral coat protein of MLBVV (kindlyprovided by Dr Y. Kawazu, National Institute of Vegetable and TeaScience, Japan).The identity of MLBVV was verified by cloning and sequencing amplicons(GenBank Accession Nos. FJ555204-05), which shared 97% identitywith both Italian (AY204674Æ1) and Brazilian (DQ854813Æ1)MLBVV isolates. Specific primers, Cps1 (5¢-CTCATGACAAAAGAAGAGAAGC) and Cpas1 (5¢-CACATCAAATATGAAGTTGTGCTC)were designed from MLBVV-RNA 3, to allow specific MLBVV detectionin RNA extracts from collected samples. The primers were tested usingsamples 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 samplingmethod was employed to collect random field samples and estimate theincidence of the disease. These were scored for symptoms and tested forinfection by RT-PCR (primers Cps1 ⁄ Cpas1). In the three lots tested, theincidence of the disease reached 60% of plants inoculated. Additionally,soil transmission experiments were conducted using healthy lettuceseedlings planted into contaminated soil or by watering with rinse waterfrom the roots of diseased field plants. After 5 weeks in a greenhouse upto 70% of the 15 seedlings inoculated with five field isolates showedbig-vein symptoms and tested positive by DAS-ELISA and RT-PCR.Three of the plants used for transmission were also positive for Lettucebig-vein associated virus (LBVaV), detected by RT-PCR using specificprimers (VP248 and VP249; Navarro et al., 2004). This is the first reportconfirming the presence of MLBVV and LBVaV infecting lettuce plants inArgentina.