First record of Alfalfa mosaic virus in Teucrium fruticans in Italy

The Teucrium genus (Lamiaceae family) contains about 300 species of evergreen and deciduous shrubs with some species widely used as ornamental plants in rock gardens. During spring 2010 and again in the same period of 2011, some plants of Teucrium fruticans L., also known as “tree germander”, growing singly in pots in a Ligurian nursery (Savona province, Northern Italy), were noted for a bright yellow calico mosaic on the leaves. About 1% of approximately 2000 plants inspected exhibited symptoms. Preliminary electron microscope observations of leaf-dips showed semispherical bacilliform particles, consistent with Alfamovirus and Oleavirus, in preparations obtained only from leaves of symptomatic plants. Three symptomatic and two asymptomatic plants were checked for the presence of Cucumber mosaic virus,or Alfalfa mosaic virus in PASELISA by using commercial kits (Bioreba, Switzerland), and Olive latent virus 2 (OLV2) by immunodecoration of virus particles with an OLV2 antiserum produced against an Italian OLV2 isolate. Symptomatic plants were positive only to AMV, whereas all asymptomatic plants were negative to all viruses checked. The virus was succesfully transmitted mechanically to Chenopodium amaranticolor and Ocimum basilicum which reacted, as expected for infections caused by AMV (1), with chlorotic local lesion followed by mosaic and bright yellow mosaic respectively. The disease was transmitted also by grafting an infected scion on healthy T. fruticans, and symptoms appeared after about 3 weeks. AMV infection in symptomatic grafted plant was verificated by PAS-ELISA, confirming that bright yellow mosaic symptoms observed in T. fruticans were induced by an isolate of AMV. Immunocapture Reverse Transcription-Polymerase Chain Reaction assay, following the protocol described by Wetzel et al. (2), was performed on leaf extracts from one symptomatic plant, using a polyclonal serum raised against a French isolate of AMV, kindly provided by H. Lot (INRA, Station de Pathologie Végétale, Avignon, France). Specific AMV primer pair was used in the RT-PCR reactions (3). A DNA fragment of c.a. 750 bp, covering the entire coat protein gene (CP), was obtained after ICRT- PCR and fragment was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega, USA), cloned and two independent clones sequenced (MWG Biotech, Germany). The consensus sequence was submitted to EMBL (Accession No. FR854391). Pairwise comparison of the AMV-T. fruticans isolate CP sequence (named Tef-1) with those of AMV reference isolates, revealed the maximum (98.0-97.3%) nucleotide identities with isolates belonging to subgroup I, 95.5-94.0% identities with subgroup IIA isolates and 95.6% identity with the subgroup IIB isolate Tec-1 (4). Among subgroup I isolates, Tef-1 had the maximum CP nucleotide identity with the CP gene belonging to an AMV isolate identified in 2010 in Lavandula stoechas in the same geographic area, suggesting a common origin for these two viral isolates. The overall results clearly indicate that an AMV isolate was the causal agent of the calico-type mosaic observed in T. fruticans. To our knowledge, this is the first report of T. fruticans as a natural host of AMV