Forest diseases : FTA cards and DNA sampling

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.