First report of orchid fleck virus and its mite vector on green cordyline
Orchid fleck virus (OFV) and its mite vector Brevipalpus californicus were for the first time identified on green cordyline plants showing distinctive chlorotic and necrotic ringspots. Thin section electron microscopy revealed bacilliform, dichorhavirus-like particles within nuclear viroplasms. RT-PCR using OFV degenerate primers yielded a single amplicon, the nucleotide sequence of which closely matched the nucleoprotein gene of OFV.
KeywordsCordyline terminalis Dichorhavirus Brevipalpus mites RT-PCR Nucleoprotein gene sequence Family Rhabdoviridae
Total RNA was extracted from both chlorotic and necrotic ringspots using RNeasy Plant Mini kit (Qiagen). Superscript One-Step RT-PCR system with Platinum Taq DNA polymerase (Thermo Fisher Scientific, Invitrogen) was used following the manufacturer’s protocol and primer pair mN2 and polydT/SP6 (Blanchfield et al. 2001). A single amplicon of about 800 bp was obtained (data not shown), similar in size to the product obtained with orchid fleck virus (OFV; species Orchid fleck dichorhavirus, genus Dichorhavirus, family Rhabdoviridae) lilyturf RNA control (Mei et al. 2016), whereas negative control samples contained no detectable bands. Amplified DNA was extracted from the PCR reaction using Wizard SV Gel and PCR clean up kit (Promega), cloned into pGEM-T Easy vector (Promega) and transformed into Omnimax E. coli cells (Thermo Fisher Scientific) following the manufacturers’ protocols. Following colony PCR, four positive recombinant clones were grown overnight and plasmid DNA was extracted for Sanger sequencing using M13 forward and reverse primers at the Australian Genome Research Facility (Brisbane). The dichorhavirus infecting green cordyline appears to be OFV, because the nucleotide sequence amplified using dichorhavirus degenerate N gene primers (GenBank accession MG812380) was 98% identical to the N gene of an OFV isolate infecting cymbidium in Japan (LC222629). The OFV isolate from cordyline was 99% identical in nucleotide sequence to the N gene fragment of Australian OFV isolates from lilyturf (KT947974) and cymbidium (KT947975) (Mei et al. 2016), indicating a close relationship between OFV isolates from various plant species in Australia.
This study identified green cordyline as a new host for OFV and for its known vector, B. californicus (Kondo et al. 2003). B. phoenicis (sensu lato) and B. obovatus have been previously found on ti plants (Miranda et al. 2007; Kitajima et al. 2010), but to our knowledge this is the first report of B. californicus mites colonizing this species. Since we did not conduct virus transmission assays with the collected Brevipalpus mites to reproduce the symptoms, thus far the mites and OFV can only be considered as associated with the disease. Infected cordyline leaf cells displayed nuclear cytopathological effects characteristic of dichorhaviruses (Kitajima et al. 2003) and typical short, bacilliform particles were seen in thin sections in viroplasms in the nucleus. No other virus-like particles were observed. So far, only cytoplasmic-type Brevipalpus-transmitted viruses have been reported for this ornamental host, associated with B. phoenicis sensu lato mites (Kitajima et al. 2010). Recently, four distinct closteroviruses, although apparently not involved in the etiology of green ti ringspot disease were identified in symptomatic plants in Hawaii (Melzer et al. 2011, 2013), and an emara-like virus has also been reported (Melzer et al. 2014), suggesting that different viruses may be associated with ringspot symptoms of cordyline in different geographical locations.
This research was jointly supported by the Queensland Department of Agriculture and Fisheries and the University of Queensland through the Queensland Alliance for Agriculture and Food Innovation. Travel and collaboration was supported by a Fapesp-UQ SPRINT grant.
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Conflicts of interest
The authors have no conflicts of interest.
Human and animal studies
This article does not contain any studies with human participants or animals performed by any of the authors.
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