New rapid PCR protocols to distinguish genetic groups in Erysiphe necator

Introduction: Genetically distinct but morphologically similar groups of Erysiphe necator Schwein. (formally Uncinula necator (Schwein.) Burrill (BRAUN and TAKAMATSU 2000) have been identified in Europe, India (DÉLYE et al. 1997) and in Australia (STUMMER et al. 2000). One group reproduces only asexually and can thus provide well-adapted persistent clones, whereas the other group can also produce recombinant genotypes through sexual reproduction. The optimal choice for control strategies thus requires the description of the distribution of both groups in vineyards. One RFLP probe or one PCR primer would probably be sufficient to identify the genetic groups because of the marked differences between them. One drawback of these techniques is the need for several preparatory steps, i.e. isolation, production of conidia and DNA extraction. To circumvent this problem, DÉLYE et al. (1999) developed nested allele-specific PCR assays based on nucleotide differences in the sequence of the gene encoding eburicol 14α-demethylase (CYP51) and in the ITS1 sequence of the rDNA. In our trials, these protocols needed to be replicated to obtain reliable results. We therefore experimented other strategies to obtain more simple and robust methods for group identification and designed specific PCR primers to target: (1) the sequences of RAPD fragments using the approach of characterized amplified region (SCAR) (PARAN and MICHELMORE 1993), and (2) a sequence identified in a genomic library enriched in microsatellite motifs. These primers were evaluated using DNA templates obtained by different methods.