QoI resistance of Plasmopara viticola in Italy: biological and quantitative Real-Time approaches.

A failure in the control of grapevine downy mildew [Plasmopara viticola (Berk & MA Curtis ex de Bary) Berl & de Toni] using azoxystrobin occurred during the year 2000 in the Emilia Romagna region in Italy. In particular, this was seen on some farms in the Ravenna district and in an experimental field of Bologna University; in the latter, a famoxadone plus cymoxanil ready mixture was also unsuccessful. In many species of pathogenic fungi, including P. viticola, the resistance phenotype to QoIs has generally been associated with a single nucleotide polymorphism (SNP) on the cytochrome bc1 mitochondrial gene that results in an amino acid change. Thus there is a single point mutation (GGT to GCT) in the cytochrome b gene, resulting in the substitution of glycine by alanine at position 143 in the gene product. To study the presence of P. viticola populations with QoI resistance, 64 P. viticola field strains were collected in vineyards in North-Eastern Italy (the Emilia Romagna, Friuli Venezia Giulia and Lombardia regions) during the three-year period from 2001-2003. These were characterised for their QoI sensitivities by biological and DNA molecular analyses. The P. viticola populations analysed by qualitative PCR using allele-specific primers for the G143A mutation showed the presence of this mutation in resistant phenotypes. To correlate the frequency of this SNP with resistant phenotypes, quantitative real-time PCR, based on the SYBR-Green I detection method, was carried out on mitochondrial DNA fragments. A comparative analysis of the quantitative PCR measurements of mutant allele frequency and phenotype (resistant or sensitive strains) showed that the QoI-sensitive populations had the mutant allele ranging from 0 to 0.3% (4 samples), while the QoI-resistant populations gave a range of mutation between 1.2 and 97.1% (60 samples). The analysis of mutant allele frequency by this quantitative PCR approach is fast and reliable, and it allows an increase in the number of samples analysed. Thus, combined with the biological assay, real-time PCR represents a valid approach in the study of the evolution of QoI sensitivity in P. viticola populations.