Development of a molecular method to detect and quantify Aphanomyces euteiches in soil

• Published in: FEMS microbiology letters Vol. 273; no. 1; pp. 64 - 69
• Main Authors: Sauvage, Hélène, Moussart, Anne, Bois, Frédéric, Tivoli, Bernard, Barray, Sylvie, Laval, Karine
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-08-2007; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Aphanomyces - genetics; Aphanomyces - isolation & purification; Aphanomyces euteiches; Bioassays; Biological and medical sciences; Colony Count, Microbial - methods; Copy number; Deoxyribonucleic acid; DNA; DNA quantification; Fundamental and applied biological sciences. Psychology; Infestation; Inoculum; Microbiology; Mycological methods and techniques used in mycology; Mycology; Oomycetes; Plant Diseases - microbiology; Plant Roots; Plasmids; Polymerase Chain Reaction - methods; real-time PCR; Root rot; Sensitivity and Specificity; soil; Soil Microbiology; Soils; Statistics as Topic; soil; real-time PCR; DNA quantification; oomycetes; Aphanomyces euteiches; Plant pathogen; Phycomycetes; Method; Real time; Fungi; Polymerase chain reaction; Soils; Detection; Thallophyta; Quantitative analysis
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2007.00784.x

A real-time PCR assay using 136F/211R primers and 161T TaqMan® probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.