Development of a PCR‐based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium‐aquaticum)

Abstract

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small-subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii , a plasmodiophorid pathogen of watercress ( Rorippa nasturtium-aquaticum ). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration-based method for obtaining pathogen DNA for PCR from watercress-bed water was developed.

Improved immunological detection of Spongospora subterranea

“…The epitope recognised could be a wall protein common in both types of cystosori. In contrast to this, both Bulman and Marshall (1998) and Bell et al (1999) did not find much homology between the DNA sequences of parts of the ITS region of each organism and the Ssn specific primers designed by Down and Clarkson (2002) failed to amplify DNA from Sss.…”

“…Most recently, van de Graaf et al (2003) used realtime PCR with newly designed primers to detect and quantify Sss in soil, water and plant tissue samples. Down and Clarkson (2002) developed a sensitive PCR-based detection system for Ssn but tested it mainly on zoospores.…”