SPECIFIC PCR BASED DETECTION OF PHYTOPHTHORA-MEDICAGINIS USING THE INTERGENIC SPACER REGION OF THE RIBOSOMAL DNA

A technique based on the polymerase chain reaction (PCR) for the speci fic detection of Phytophthora medicaginis was developed using nucleoti de sequence information of the ribosomal DNA (rDNA) regions. The compl ete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and r elated species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 b p) within the IGS 2 of P. medicaginis using the PCR, was designed. Spe cific amplification of this fragment from P. medicaginis was highly se nsitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000:1. Specific PCR amplification using PPED04 and PP ED05 was successful in detecting P. medicaginis in lucerne stems infec ted under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identif ication and detection of a wide range of Phytophthora spp. in plants a nd soil.