Abstract:
Potyvirus is the largest and one of the most economically important of the virus genera infecting
plants. The complexities of potyvirus identification resulting from many different species, mixed
infections, emerging new viruses, new hosts, and new vectors, etc., often requires the use of
multiple detection methods which is time consumable and costly. Therefore an assay that can test
for a range of potyviruses simultaneously, with good specificity and sensitivity, is desirable. This
study looked at the feasibility of producing an oligonucleotide microarray for detection and
identification of potyviruses at both species and strain level.
Thirty plant samples with suspected potyvirus infections were collected from field and research
laboratories in New Zealand and partial NIb gene, complete CP gene and 3΄UTR were sequenced.
Twelve definitive potyviruses, one tentative potyvirus, one non-potyvirus, and one novel potyviruslike
sequence were identified, six of which were first records for New Zealand.
Sequence analysis showed that NIb and CP genes and the 3΄UTR contained both conserved and
variable sequences which were used to design both species and strain level specific probes. Four
Potyvirus species were chosen for a “proof of concept” study and probes were designed using two
different software programs (ROSO and CAG software). A total of eighty five probes including 33
perfect-match and 52 mismatch probes were selected to represent the four targeted potyviruses.
Each probe was synthesized with spacers of either 6 or 12 poly-cytosine or poly-thymine at the 5΄
terminus. Arrays showed high specificity to the targets when tested using nineteen different
geographically diverse potyvirus isolates representing the four target species, four distinct but
closely related New Zealand potyviruses, and four healthy plant species. Factors affecting the
hybridization efficiency, e.g. the size of the target fragments, secondary structure of probes and
targets, label type, strandedness, Tm and GC content of probes, were also investigated.
The approaches and protocols developed in this study should form a useful basis for developing
other potyviruses arrays and the results also provide useful insights into issues of generic interest
for the development of arrays for detecting other pathogens.