First Report of Alfalfa mosaic virus Infecting Lavandula × intermedia in Croatia.

Lavandin (Lavandula × intermedia Emeric ex Loiseleur) is cultivated on a large scale in some South European countries for the extraction of essential oils or as an ornamental plant for gardens and landscapes. In May of 2012, virus-like symptoms including bright yellow calico mosaic, leaf distortion, and growth reduction were observed on 15% of lavandin plants in a commercial nursery in Banovo Brdo locality, Baranja County, Republic of Croatia. Leaves from 15 symptomatic lavandin plants were collected and examined by double-antibody sandwich (DAS)-ELISA using commercial antisera (Bioreba AG, Reinach, Switzerland) against two viruses known to infect Lavandula spp.: Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) (2,3). Commercial positive and negative controls and extracts from healthy lavandin leaves were included in each ELISA. Only AMV was detected serologically in all 15 tested samples. Five plants each of Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana were mechanically inoculated with sap from an ELISA-positive sample (70-12) using 0.01 M phosphate buffer (pH 7). Local chlorotic spots accompanied by systemic mosaic on both Chenopodium species and bright yellow mosaic on N. benthamiana were observed 6 and 12 days post-inoculation, respectively. Test plants were assayed by DAS-ELISA and all inoculated plants of each species tested positive for AMV. The presence of AMV in all symptomatic lavandin plants was further confirmed by reverse transcription (RT)-PCR assay. Total nucleic acid was extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using AMV specific primer pair CP AMV1 (5'-TCCATCATGAGTTCTTCAC-3') and CP AMV2 (5'-AGGACTTCATACCTTGACC-3') (1). Total RNAs obtained from the Serbian AMV isolate from alfalfa (GenBank Accession No. FJ527748) and healthy L. × intermedia plant served as the positive and negative control, respectively. The 751-bp amplicons, covering the partial coat protein (CP) gene and 3'-UTR, were obtained from all 15 samples that were serologically positive to AMV as well as from positive control. No amplification product was observed when extract from healthy L. × intermedia plant was used as template in the RT-PCR assay. The RT-PCR product derived from isolate 70-12 was directly sequenced in both directions using the same primer pair as in RT-PCR and deposited in GenBank (JX996119). Multiple sequence alignment of the CP open reading frame was performed by MEGA5 software (4) and revealed that the isolate 70-12 showed the highest nucleotide identity of 99.4% (99.5% amino acid identity) with Serbian AMV isolate from tobacco (FJ527749). To our knowledge, this is the first report of AMV on L. × intermedia in Croatia. Because lavandin is an aromatic plant traditionally and widely grown in Croatia, the presence of AMV could be a limiting factor for its successful production. References: (1) M. M. Finetti-Sialer et al. J. Plant Pathol. 79:115, 1997. (2) T. Kobylko et al. Plant Dis. 92:978, 2008. (3) L. Martínez-Priego et al. Plant Dis. 88:908, 2004. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.