HIGHLY SPECIFIC AND SENSITIVE NONRADIOACTIVE MOLECULAR-IDENTIFICATIONOF PHYTOPHTHORA-CINNAMOMI

In response to the need for a faster, more reliable method for identif ying Phytophthora cinnamomi in cork oak soils in Portugal, a simple, f ast, sensitive molecular identification method is described. It is bas ed on a colorimetric assay which involves an oligonucleotide capture p robe covalently immobilised on microtitration wells, a multi-biotinyla ted oligonucleotide detection probe and the PCR-amplified target DNA. The target DNA is a 349 bp DNA fragment partially covering the 3'-tran slated and 3'-untranslated regions of the cinnamomin gene. When the sp ecificity of the PCR reaction was evaluated in vitro using isolates of P. cinnamomi and eight other Phytophthora species, including the rela ted P. cambivora, it was specific to P. cinnamomi. When 30 isolates of P. cinnamomi from oak roots in southern Portugal were assayed, 26 gav e a strong positive response. The assay has a sensitivity of about 2-5 genome equivalents of P. cinnamomi. The reason for the negative respo nse of four isolates remains unclear.