First Record of Alfalfa mosaic virus in Teucrium fruticans in Italy

The Teucrium genus (Lamiaceae family) contains ~300 species of evergreen and deciduous shrubs with some species widely used as ornamental plants in rock gardens. During the springs of 2010 and 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 (~1% of ~2,000 plants inspected exhibited symptoms). Preliminary electron microscope observations of leaf-dips showed semispherical to 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 Cucumber mosaic virus or Alfalfa mosaic virus (AMV) in protein A sandwich (PAS)-ELISA with commercial kits (Bioreba, Reinach, 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 and all asymptomatic plants were negative to all viruses checked. The virus was successfully transmitted mechanically to Chenopodium amaranticolor and Ocimum basilicum that reacted, as expected for infections caused by AMV (1), with a 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. Symptoms appeared after ~3 weeks in one plant of six grafted. AMV infection in a symptomatic grafted plant was verified by PAS-ELISA, confirming that bright yellow mosaic symptoms observed in T. fruticans were induced by an isolate of AMV. Immunocapture reverse transcription (IC-RT)-PCR assay, following the protocol described by Wetzel et al. (4), was performed on leaf extracts from one symptomatic plant using a polyclonal serum raised against a French isolate of AMV, provided by H. Lot (INRA, Station de Pathologie Végétale, Avignon, France). Specific AMV primer pair was used in the RT-PCR reactions (2). A DNA fragment of ~750 bp, covering the entire coat protein gene (CP), was obtained after IC-RT-PCR. The amplicon was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI), cloned into pGEMT-easy vector (Promega) and two independent clones sequenced on both strands at MWG Biotech (Ebersberg, Germany). The consensus sequence was submitted to EMBL (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 to 97.3%) nucleotide identities with isolates belonging to subgroup I, 95.5 to 94.0% identities with subgroup IIA isolates, and 95.6% identity with the subgroup IIB isolate Tec-1 (3). 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. 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. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. +Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Arch. Virol. 156:1049, 2011. (4) T. Wetzel et al. J. Virol. Methods 39:27, 1992.