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Specific PCR based detection of Phytophthora medicaginis using the intergenic spacer region of the ribosomal DNA

Published online by Cambridge University Press:  01 January 1998

E. C. Y. LIEW ,
D. J. MACLEAN  and
J. A. G. IRWIN
E. C. Y. LIEW
Affiliation:
Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Qld 4072, Australia
D. J. MACLEAN
Affiliation:
Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Qld 4072, Australia
J. A. G. IRWIN
Affiliation:
Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Qld 4072, Australia
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Abstract

A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete 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 related 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 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000: 1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

Type
Research Article
Information
Mycological Research , Volume 102 , Issue 1 , January 1998 , pp. 73 - 80
Copyright
© The British Mycological Society 1998

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