First Report of Orchid fleck virus in Lilyturf (Liriope spicata) in Australia

Liriope spicata (Asparagaceae) is an evergreen ornamental plant commonly referred to as lilyturf, creeping liriope, monkey grass, or creeping lilyturf, and is widely grown as groundcover and for erosion control (Fantz 2008). In April 2015, lilyturf plants with bright yellow flecks on the leaves were observed in landscape plantings across the St. Lucia campus of the University of Queensland, Brisbane, Australia. These symptoms were reminiscent of those observed on leaves of orchid species infected by Orchid fleck virus (OFV) (Kubo et al. 2009). Symptomatic leaf samples were collected and analyzed in a JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan). Leaf dips were prepared by breaking up symptomatic tissue in 1% ammonium molybdate, pH 7.0 negative stain and extracts were placed on nitrocellulose-coated copper grids. Digital micrographs were captured with an Orius digital camera (Gatan Inc., Pleasanton, CA). Typical nonenveloped, rhabdovirus-like, bacilliform particles measuring ∼45 × 95 nm were observed, similar to those of OFV from infected orchids (Kondo et al. 2006). Total RNA was extracted from symptomatic tissue using an RNeasy Plant Mini Kit (Qiagen) and subjected to RT-PCR using the Superscript III One Step RT-PCR System with Platinum Taq DNA Polymerase (Thermo Fisher) and OFV nucleoprotein (N) gene primers polydT/SP6 (5′-GATTTAGGTGACACTATAGTTTTTTTTTTTTTTTTT(A/G/C)-3′) and mN2 (5′-TGCAGGAATATAGCCGACATGTT-3′) (Blanchfield et al. 2001). Agarose gel electrophoresis showed a single amplicon of ∼800 bp, comparable to the OFV positive control. DNA from both the lilyturf (DAF Plant Virus Collection Accession No. 5216) and a cymbidium OFV control (Accession No. 703) amplicons were gel-purified and cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced using dideoxynucleotide sequencing at the Australian Genome Research Facility (Brisbane, Australia). A GenBank BLASTx search of the lilyturf virus N gene sequence of 658 nt showed 100% match to the N protein sequence of an Australian OFV isolate (Genbank Accession No. AAK09392 and e-value 1e). The partial N gene nucleotide sequence of the lilyturf OFV isolate #5216 was 99% identical to the cymbidium OFV isolate. Both sequences have been deposited in GenBank with Accession Nos. KT947974 and KT947975, respectively. Based on the observed symptoms, particle morphology, N gene amplicon using OFV primers, and high sequence identity with other OFV sequences, we conclude that the lilyturf plants were infected with OFV. This is the first report of OFV infecting L. spicata. This alternative host may play a possible role in OFV spread to orchids or other ornamental species that are colonized by its Brevipalpus sp. mite vector