First report of lettuce big-vein virus and Mirafiori lettuce virus in Chile

Lettuce big-vein disease (BVD) is a serious virus disease of lettuce. Recent evidence has brought into question the role of Lettuce big-vein virus (LBVV) in the etiology of BVD, and suggested that Mirafiori lettuce virus (MiLV) and not LBVV is the causal agent of BVD (1,2). Lettuce plants (Lactuca sativa L.) with symptoms similar to those of BVD were observed during the winter of 2003 in open-field and hydroponic-grown lettuce plants located in the Chacabuco Province of central Chile. Symptomatic plants exhibited leaves with chlorotic vein banding that became ruffled and distorted. Symptoms were usually accompanied by reduced plant size and absence of head formation. Roots from symptomatic plants were analyzed by light microscopy-acid fuchsin staining. Zoosporangia and resting spores of Olpidium brassicae were identified on the basis of their morphology and structure. Additionally, soil transmission experiments were performed with 50 healthy lettuce seedlings replanted into contaminated soil collected from lettuce fields having symptomatic crops. After 3 weeks, one-half of the seedlings showed differing degrees of big-vein symptoms, and the presence of spores of O. brassicae was confirmed in the roots by light microscopy. Seedlings raised in sterilized soil showed no symptoms after the same period of time. On the basis of nucleotide sequences of LBVV and MiLV from the GenBank database, primers specific to the coat protein genes of each virus were designed as follows: MiLVV-CP1: 5'-CAAATCTGTCCACAATTCC-3'; MiLVV-CP2: 5'-TCTCACTTGAAAACCTTCC-3'; MiLVV-CP3: 5'-TTGCAACGTGATGAAACC-3'; MiLVV-CP4: 5'-AAAGAAGAGAAGCCTGTTCC-3'; LBVV-CP1: 5'-AAGCTTTCCGTACTGTCC-3'; LBVV-CP2: 5'-CCTTGATACAGTTTTTGACC-3'; LBVV-CP3: 5'-GTATGCTGATTTCTGTAGACC-3'; LBVV-CP4: 5'-TAGATGTTCTTCGCCACC-3'. The primers were used in reverse transcription-polymerase chain reaction (RT-PCR) assays with dsRNA as a template. Amplicons of the expected size were obtained in each of the five symptomatic plants analyzed, using each of the designated primer sets: MiLVV-CP1/MiLVV-CP2: 562 bp; MiLVV-CP3/MiLVV-CP4: 743 bp; LBVV-CP1/LBVV-CP2: 485 bp; and LBVV-CP3/ LBVV-CP4: 570 bp. No amplicons were obtained from healthy lettuce plants. The identity of both viruses was verified by cloning and sequencing of the amplicons. Nucleotide sequences were compared with those in the GenBank database. Sequences derived from the Chilean isolates resulted in identities of 87 to 97% for MiLV and 97 to 99% for LBVV. All samples a