3.3.3S
< b >WHAT ARE THE INGREDIENTS OF A SUCCESSFUL DNA BASED DIAGNOSTIC TEST FOR FUNGAL PLANT PATHOGENS < /b >
< b >WHAT ARE THE INGREDIENTS OF A SUCCESSFUL DNA BASED DIAGNOSTIC TEST FOR FUNGAL PLANT PATHOGENS < /b > ANDRE DRENTH and GABRIELE WAGELS Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland 4072 Brisbane, Australia < b > Background and objectives < /b > < i > Phytophthora < /i > incited diseases cause severe devastation to a great variety of tree, ornamental, and crop plant species. Disease free planting material and disease free soil is of vital importance in the production of healthy plants. The objective of our project was to design a DNA based diagnostic test for a number of species of the genus < i > Phytophthora < /i > which is quick, reliable, sensitive and highly specific as well as amenable to routine screening of plant material and soil for the presence of < i > Phytophthora < i > . < b > Results and conclusion < /b > In order to design successful PCR based diagnostic tests background information concerning the population and evolutionary biology of the organisms under investigation is needed. The following is a systematic approach we used to develop PCR based diagnostics for a number of < i > Phytophthora < /i > species. (1) Assess levels of genetic diversity present in targeted < i > Phytophthora < /i > species and select authentic and representative isolates from each species. (2) Obtain RDNA sequence information from representative strains of each of the species (3) Reveal evolutionary relationships among the different < i > Phytophthora < /i > species based on RDNA sequence information and relate this to relevant biological species (4) Use RDNA sequence information to design and synthesise PCR primers which are highly specific for each of the targeted species. (5) Test primers under predetemiined testing conditions to reveal the most optimal primer combinations for effective amplification of the RDNA target area. (6) Test primers for specificity on predetermined tester series consisting of < i > Phytophthora < /i > species, other root pathogens, endophytes and symbiotic bacteria under standard conditions. (7) Test sensitivity of highly specific primer combinations. (8) Assessment of the reliability and sensitivity of the PCR based diagnostic test compared to direct isolation from plant tissue, and to conventional bait testing for < i > Phytophthora < /i > from soil. (9) Optin-iise a DNA based diagnostic test for routine use. In selecting DNA sequences useful as a target for detection of the genus < i > Phytophthora < /I> and species within this genus it is important to find sequences which are highly stable. There must be sufficient variation between different species to allow species-specific detection, and sufficiently conserved areas within species to allow detection of all isolates within any given species or within the genus as a whole. Genes encoding ribosomal RNA in eukaryotic organisms possess these qualities and we have used these sequences in the above approach to successfully design genus and five species specific primer combinations for rapid detection and identification of < i > Phytophthora < /i > .