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Forest diseases : FTA cards and DNA sampling

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Sulistyawati, P (2010) Forest diseases : FTA cards and DNA sampling. Research Master thesis, University of Tasmania.

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Abstract

The identification of plant pathogens often requires rapid, effective and reliable
sampling techniques for pathogens or -infected plant tissues. The first and
critical step to a PCR-based identification from the processing of sampled
tissues is the extraction and purification of template DNA of suitable quality
for PCR. Many DNA extraction techniques for plant and fungal DNA are time consuming
and/or require sophisticated laboratory equipment. Whatman
International Ltd from Flinders Technology Associates (FTA) has patented
cards which offer a simple and rapid method for the room temperature
collection, transport and storage (short and long term) of DNA. Direct capture
of plant pathogen DNA in the field, achieved by squashing infected tissue
(either symptomatic or asymptomatic) and/or pathogen structures onto cards,
will facilitate the detection and identification o~ the pathogen. DNA sampling
with these cards provides many advantages for a plant pathologist such as
increasing the number of samples that can be collected, stored and transported
in the field, especially in remote locations. It circumvents any requirement for
travelling with collection containers, cumbersome equipment or labile buffers.
It is particularly useful when the isolation of a pathogen is either not possible,
as for an obligate pathogen, or only achieved with a low rate of success.
The aims of the research in this thesis were to investigate the applicability of
FTA cards as a new method for DNA sampling from fungal and infected plant
material associated with forest diseases. After DNA sampling and capture on
the card, the DNA extractions were subjected to PCR, DNA sequencing and
species-specific PCR. There were three main sources of material squashed onto
the FTA cards; fungal material (cultures, fruitbodies and spores); asymptomatic
or symptomatic plant material (root, leaves, and seeds); water and soil that
were likely to contain infective propagules.
DNA was easily obtained from fungal cultures squashed onto cards and the
DNA thus harvested is suitable for PCR, DNA sequencing and species specific
PCR. With the latter type of PCR, caution must be exercised when
using the card in order to avoid contamination. In case studies of forest
diseases involving the squashing of infected material onto FTA cards, several
fungal pathogens were identified based on sequencing of the PCR product
obtained from the DNA captured by the FTA card; Fusarium oxysporum,
Cylindrocladium spp., Phoma spp., and Phytophthora spp. were detected and
identified. The use of FTA cards to sample the DNA of certain fungal
propagules such as rust spores or the fungal propagules contained in soil or
water did not prove very successful.
These preliminary results clearly demonstrate the potential of FTA cards to
assist forest pathologists in disease detection and identification. Further
modifications to FTA card sampling techniques are discussed so that the DNA
of a wide range of forest pathogens can be successfully obtained from plant
tissue, soil or water.

Item Type: Thesis (Research Master)
Copyright Holders: The Author
Copyright Information:

Copyright 2010 the author

Additional Information:

Available for use in the Library and copying in accordance with the Copyright Act 1968, as amended. Thesis (MAgrSc)--University of Tasmania, 2010. Includes bibliographical references

Date Deposited: 04 Feb 2015 23:37
Last Modified: 24 Jun 2016 04:49
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