A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus E. Yergeau, M. Filion 1 , V. Vujanovic, M. St-Arnaud * Institut de recherche en biologie ve ´ge ´tale, Universite ´ de Montre ´al and Jardin botanique de Montre ´al, 4101 East, Sherbrooke Street, Montre ´al, QC, Canada H1X 2B2 Received 8 April 2004; received in revised form 9 September 2004; accepted 9 September 2004 Available online 12 October 2004 Abstract In North America, asparagus (Asparagus officinalis ) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani , clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples. D 2004 Elsevier B.V. All rights reserved. Keywords: Fusarium; PCR-DGGE; Asparagus officinalis ; Crown and root rot; Elongation factor 1 alpha; Fingerprinting; Fungal biodiversity 1. Introduction Asparagus (Asparagus officinalis ) is a low-input perennial crop having a high market value. Yet, asparagus fields are frequently showing a marked 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.09.006 * Corresponding author. Tel.: +1 514 872 1439; fax: +1 514 872 9406. E-mail address: marc.st-arnaud@umontreal.ca (M. St-Arnaud). 1 Present address: Department of Biology, Universite ´ de Moncton, Moncton, NB, Canada E1A 3E9. Journal of Microbiological Methods 60 (2005) 143 – 154 www.elsevier.com/locate/jmicmeth