Analysis of the distribution of Phytophthora cinnamomi in soil at a disease site in Western Australia using nested PCR

The oomycete plant pathogen Phytophthora cinnamomi has infected a very large area of native vegetation in the south western corner of Australia. An important aspect of effective disease management depends on being able to accurately map areas of infestation. For this purpose, we have developed a nested polymerase chain reaction (PCR) protocol for the detection of P. cinnamomi in soil. The test uses two sets of primers developed from the rRNA ITS sequences of P. cinnamomi and can detect as little as 1 pg DNA. The degree of sensitivity was reduced with DNA extracted from soil although this depended on the type of soil. Soils with a high organic content, such as eucalypt forest soil and potting mix were more inhibitory than sandy soils. Inhibition by soil DNA could be reduced by the addition of bovine serum albumin and formamide to the reaction. Taq DNA polymerase was very sensitive to inhibitors compared with Tth(+) or TaqF1*. In comparison with baiting (0-10% positive samples), nested PCR proved to be a very much more efficient (90-100% positive samples) method for the detection of P. cinnamomi in soil.