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Detection and molecular characterisation of Orchid Fleck Virus


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Blanchfield, AL 2000 , 'Detection and molecular characterisation of Orchid Fleck Virus', Research Master thesis, University of Tasmania.

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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
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.

Item Type: Thesis - Research Master
Authors/Creators:Blanchfield, AL
Keywords: orchids, diseases and pests of orchids, plant Rhabdovirus group
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