Nested allele-specific PCR primers distinguish genetic groups of Uncinula necator

Isolates of the obligately biotrophic fungus Uncinula necator cluster in th ree distinct genetic groups (groups I, II, and III). We designed PCR primer s specific for these groups in order to monitor field populations of U. nec ator. We used the nucleotide sequences of the gene that encodes eburicol 14 alpha-demethylase (CYP51) and of the ribosomal DNA internal transcribed sp acer 1 (ITS1), ITS2, and 5.8S regions. We identified four point mutations ( three in CYP51 and one in ITS1) that distinguished groups I and II from gro up III based on a sample of 132 single-spore isolates originating from Euro pe, Tunisia, Israel, India, and Australia. We developed a nested allele-spe cific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from v ineyards. In a preliminary study performed with samples from French vineyar ds in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolate s to a population composed primarily of group III isolates occurred during the grapevine growing season.