3.3.15
DETECTION OF FUNGAL PHYTOPATHOGENS BY PCR

PA O'BRIEN1, MD FARBEY1 and KJ NOWAK2

1School of Biological Sciences and Biotechnology, Murdoch University, Perth, WA 6152 Australia; 2Australian Neuormuscular Research Inst., QEII Medical Centre, Nedlands, WA 6009 Australia

Background and objectives
Methods used for the identification of pathogenic fungi are tedious, time consuming, highly error prone and require a high degree of technical expertise. Detection of fungi in material such as soil involves plating on selective media or baiting, both of which may favour those isolates which are faster growing, and consequently do not yield a representative picture of the fungal population. An alternative approach is based on detection of DNA sequences specific to the pathogen using the polymerase chain reaction (PCR) [1]. However this requires the ability to isolate DNA in a form in which it can be amplified. Phenolic and other compounds, e.g., polysaccharides present in soil, or in many plant species inhibit amplification [2]. Although techniques for extraction of PCR amplifiable DNA from soil have been developed for bacterial pathogens, they are too complex for studies with large numbers of samples. What is needed is a simple procedure which results in the extraction of PCR amplifiable DNA from soil or plant material. We have investigated conditions for extraction of amplifiable DNA from soil.

Materials and methods
DNA was extracted from Phytophthora cinnamomi and amplified using the ITS 1 and ITS 2 primers as described [3]. The presence of inhibitors in extracts was assayed by addition of extracts to PCR reactions containing Pcinnamomi DNA.

Results and conclusions
Extracts prepared by boiling mycelium (amounts of 1mg or less) significantly reduced the amount of PCR product obtained by amplification of P. cinnamomi DNA with the ITS 1 and 2 primers. The reduction in the amount of product was proportional to the volume of extract added. Addition of extracts prepared by boiling mycelium and soil (1g) mixtures, or soil alone completely inhibited amplification. Purification of extracts by chromatography on Sephadex, or with a number of other commercially available DNA purification systems appeared to remove inhibition of amplification, however when these extracts were concentrated, inhibition returned. Less inhibition was observed when the extracts were diluted with water. The inclusion of repeated freeze-thaw cycles in the extraction procedures did not enhance amplification. The inclusion of PVP or PVPP in the extraction buffer significantly reduced the degree of inhibition. Further reduction in inhibition was achieved by the inclusion of spermidine and BSA in the PCR reaction. Detection of products by use of labelled probe increased the sensitivity of the PCR detection 10 fold. Attempts to increase the sensitivity of detection by scaling up the reaction for use with samples greater than 1 g soil were unsuccessful. Extracts prepared from larger samples (5-50g) when concentrated, always inhibited amplification. This could not be overcome by prior separation of the mycelium from the soil using magnetic beads crosslinked to anti-mycelium antibodies. In comparative studies the PCR test allowed detection of P. cinnmaomi in soil samples in 24 hr, whereas the baiting procedure took 10 days. In addition the PCR test was 10 fold more sensitive. The results of further studies between PCR detection and traditional tests will be presented.

References
1. Henson, J.M. and French, R. (1993) Ann Rev Phytopathol 31, 81-109.
2. Young, C.C. et al., (1993) Appl. and Environmental. Microbiol. 59, 1972-1974.
3. Bruns, T.D., White, T.J. and Taylor, J.W. (1991) Ann Rev. Ecol. Syst. 22, 525-564.