Development of Diagnostic Tools for the Seed Potato Industry
Mortimer-Jones, Sheila Mary (2010) Development of Diagnostic Tools for the Seed Potato Industry. PhD thesis, Murdoch University.
The Australian potato industry is threatened by inadequate measures to control the virus health of seed potatoes. Potatoes are vegetatively propagated; therefore infection can result in disease spreading through generations. This has the potential to cause significant economic losses. Virus testing on tuber sprouts is currently conducted by ELISA, however a significant time delay of several weeks can occur while tubers sprout. There is a considerable need for a rapid, quantitative and cost effective virus test directly on bulked samples of dormant tubers to identify infected lots during seed multiplication.
The potato viruses of economic importance in Western Australia are Potato virus S, (PVS), Potato virus X, (PVX), Potato leafroll virus, (PLRV) and Tomato spotted wilt virus (TSWV). The main aim of this project was to develop reliable PCR-based methods for multiplex real-time quantitative detection of these viruses in bulked potato tuber samples for seed certification for domestic and export markets.
Knowledge of the distribution of the viruses within tuber tissue was needed to develop more effective methods of RNA extraction. The distribution of the viruses in histological sections of potato tubers was investigated using immunohistochemistry and in situ hybridization. All four viruses were found to be distributed at the stolon end of freshly harvested infected potato tubers. Extraction of RNA from tuber tissue is problematic because it contains starches and phenolics which inhibit RT-PCR. Extracting RNA from the tuber peelings would overcome this problem; however one of the viruses, PLRV, is restricted to the phloem in potato tubers. The distribution of the phloem in the superficial tissue of potato tubers was therefore investigated using histological staining and transmission electron microscopy. The vascular tissue was found to be within 2 mm below the epidermis of the tuber. With this knowledge, total RNA was extracted rapidly and efficiently from bulked potato peelings equivalent to 300 dormant tubers to detect single infections of PLRV, PVX, PVS and TSWV.
For the quantitative detection of these viruses in potato leaves and tuber tissue, specific primers and fluorescent-labeled TaqMan® probes were designed. A realtime multiplex, single tube RT-PCR assay was developed. The main tasks involved primer design, optimization of reagents, standardization of RNA samples from which standard curves for analysis were generated, and identification of a baseline on which to interpret results.
Limits of detection sensitivity were established using a range of virus transcript copy numbers (8 x 101 to 8 x 109 copies of PVX and PVS, 1 x 102 to 1 x 1010 copies of PLRV and 1 x 103 to 1 x 1010 copies of TSWV). The multiplexed assay was validated in blind studies. This high-throughput test is accurate and sensitive, and as a result this test is in the process of being commercialized and used by the seed potato industry of Western Australia as a cost-effective diagnostic tool to detect viruses reliably in bulked samples of dormant potato tubers.
|Publication Type:||Thesis (PhD)|
|Murdoch Affiliation:||School of Biological Sciences and Biotechnology|
|Supervisor:||Jones, Michael, Jones, Roger and Dwyer, Geoff|
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