whole-blanchfield-thesis.pdf (4.83 MB)
Detection and molecular characterisation of Orchid Fleck Virus
thesisposted on 2023-05-26, 03:01 authored by Blanchfield, AL
Orchid fleck virus (OFV) is one of three commonly found viruses infecting orchids. Infected plants have reduced flower quality and often unsightly leaf markings and are unsaleable. Effective disease control relies on routine detection of the virus, but foliar symptoms can vary markedly and are often not reliable for diagnosis. Current laboratory tests use examination of leaf sap by electron microscopy. This is time consuming and costly. The preferred immunological tests are not available as OFV has an unstable virion and attempts to purify virus particles for antibody production have failed. However, direct isolation of viral nucleic acid from infected plants may enable development of alternative detection systems. In this project OFV was characterised by mechanical inoculation to alternate hosts. Sap inoculation was found to be difficult and affected by glasshouse temperatures, however, was successful at temperatures significantly lower than previously reported. The host range of a Tasmanian isolate of OFV was found to be different to that previously reported for isolates of OFV from Japan. Attempts to purify OFV and clone the viral RNA were unsuccessful, however primers specific to OFV were obtained. OFV was detected using RT-PCR with a primer complementary to a region of its nucleoprotein gene together with a polydT/SP6 primer complementary to the 3' terminus of the genomic segment. The resulting DNA fragments were 0.8kb long and their sequences were determined directly. The sequences of DNA fragments obtained from 33 OFV isolates from Australia, Brazil, Germany and South Africa were shown to be closely related (<2.5% difference), but a single German isolate was clearly a distinct strain and the . sequence of the targeted region of its nucleoprotein gene differed from that of the others by about 16%. Failure of RT-PCR using a second primer set complimentary to part of the phosphoprotein gene of the Japanese OFV isolate with all OFV isolates tested suggests that the Japanese isolate may represent a third distinct strain of OFV or a different virus. A search of the international nucleotide database with the OFV sequences showed them to be related, but distantly, to regions of the genomes of three plant rhabdoviruses. Isolates of coffee ringspot virus (CoRSV), citrus leprosis virus (CiL V), a common violet (Viola sp.), schefflera, hibiscus, ivy and ligustrum leaves showing ringspot symptoms and containing small bacilliform virus particles were tested using RT-PCR and the OFV specific primers. A single product of 800bp was amplified from one isolate of coffee ringspot virus and the violet sample using the primer complementary to a region of the OFV nucleoprotein gene together with a polydT/SP6 primer. The DNA products were shown to be identical to OFV when sequenced. No other sample gave an amplified product. These results suggest citrus leprosis disease, ligustrum ringspot and the ringspots on schefflera, hibiscus and ivy are caused by viruses different to OFV. However, this study was completed with a limited number of samples and the results are not conclusive, the relationship between these viruses should be further investigated.
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