Pathological and molecular characterisation of lettuce big-vein virus and associated virus from Egypt

Lettuce (Lactuca sativa) is known to be attacked by many viruses causing disease epidemics among which Lettuce big-vein virus (s) is (are) shown here to cause considerable quality and yield losses. In winter 2014, samples suspected of being infected with lettuce big-vein disease viruses (Mirafiori lettuce big-vein virus, MLBVV and Lettuce big-vein associated virus, LBVaV) were collected from naturally infected plants grown at different locations, from two governorates in Egypt (El-Qalyubia and El-Fayoum). The characteristic symptom of the disease was seen as the development of a clear region along the major veins of leaf due to the disappearance of chlorophyll making the veins appear as if they were bigger than normal. Along with this symptom, leaves were crinkled and head size was reduced and assumed elongated shape. Three isolates (two of MLBVV, 1 and 2 and one of LBVaV) were detected in lettuce specimens giving negative reaction to cucumber mosaic virus (CMV) and lettuce mosaic virus(LMV)to specific polyclonal antisera using DAS-ELISA. Detection was based on the RT-PCR technique with a pair of specific primers for coat protein gene of both MLBVV and LBVaV as it resulted in the generation of a unique PCR amplification product of approximately 233 bp for MLBVV and approximately 360bp for LBVaV. It was also proven that MLBVV and LBVaV isolated here are transmissible by the fungal vector Olpidium virulentus. The fungus was isolated from plant roots and seeds andits identity was confirmed using the PCR technique. The length of the product amplified was approximately 632 bp using a specific pair of primers for the fungal ITS region. Purification of MLBVV and LBVaV was achieved and the two viruses were detectable and distinguishable by polymerase chain reaction (PCR) and electron microscopy. Samples with symptoms that were positive by RT-PCR to MLBVV and LBVaV were gel-purified and sequenced using specific forward and reverse primers. Sequences related to the three virus isolates (MLBVV1, MLBVV2 and LBVaV1) were submitted to Genbank database under the accession numbers, LT721898, LT721899 and LT721900. The highest similarity percentage was observed between MLBVV1 and MLBVV2 and a Spain isolate was 70.3% and 69.7%, respectively. While, the highest similarity percentage between LBVaV1 and Brazil isolate was 100%.To the best of our knowledge this is the first report of lettuce big-vein disease in Egypt أظهرت الملاحظات الحقلية التي أجريت أن أكثر الاعراض شيوعا على