First Record and Complete Nucleotide Sequence of Alfalfa mosaic virus from Lavandula stoechas in Italy

During spring 2009, lavender plants (Lavandula stoechas L.) showing a bright yellow mosaic of calico type and light stunting were observed in a commercial nursery in Liguria Province in northern Italy. Of 300 plants inspected, ~2% were symptomatic. Preliminary observations of leaf sap with the transmission electron microscope revealed bacilliform virus-like particles in three symptomatic plants, whereas no virus-like particles were observed in asymptomatic plants. The same symptomatic plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, and Alfalfa mosaic virus (AMV). All three plants reacted positively against AMV antibodies, but not the other antibodies. A crude sap extract obtained from a single symptomatic plant, hereafter referred to as the Lst isolate, was prepared by macerating 1 g of fresh leaves in 4 ml of sodium phosphate 0.03 M, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Sap extract was mechanically inoculated onto a set of herbaceous hosts. Inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in bean (Phaseolus vulgaris L., cv. Borlotto) and cowpea (Vigna unguiculata L., cv. Black eye); necrotic local lesions followed by systemic necrosis in broad bean (Vicia faba L., cv. Super Simonia) and tomato (Solanum lycopersicum L., cv. San Marzano); and bright yellow mosaic (calico type) in basil (Ocimum basilicum L., cv. Gigante). To sequence the entire genome of the Lst isolate, total RNA was extracted from infected samples with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to AMV-specific reverse transcription-PCR by using four primer pairs for each genomic RNA of overlapping oligonucleotides according to the complete genome sequence of AMV 425L isolate (GenBank No. L00163 for RNA1, X01572 for RNA2, and K03542 for RNA3). The 5'- and 3'-terminals regions of each RNA were amplified with the strategy described by Lozano et al. (1) and specific AMV oligonucleotides designed for the corresponding viral RNA. The complete genome of the AMV-Lst isolate comprised 3,643 nucleotides for RNA1 (No. FN667965), 2,593 nucleotides for RNA2 (No. FN667966), and 2,038 nucleotides for RNA3 (No. FN667967). Comparative sequence analyses revealed that the AMV-Lst isolate from Italy shared overall nucleotide sequence identities with the AMV isolate 425L of 97.1, 95.5, and 94.7% for RNA1, 2, and 3, respectively. P1 and P2 replicase genes and the movement protein and coat protein (CP) genes of AMV-Lst isolate showed, respectively, 97.2, 95.1, 96.2, and 97.8% identity with those from the 425L isolate. The AMV-Lst CP gene was shorter by nine nucleotides compared with the CP gene of 425L. A phylogenetic tree, obtained with neighbor-joining and maximum likelihood methods, showed that the Lst isolate grouped within subgroup I of AMV isolates confirmed that the differences between subgroups I and II correlate mainly with the geographic origin of isolates (2). Lst represents the first Italian isolate of AMV completely sequenced, and to our knowledge, this is the first report of this virus in L. stoechas. References: (1) G. Lozano et al. Arch. Virol. 151:581, 2006. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000.