Real‐time PCR Assay with SNP‐specific Primers for the Detection of a G143A Mutation Level in Venturia inaequalis Field Populations

Fourteen primers were designed specific to SNP (G→C mutation) in the cytochrome b gene of Venturia inaequalis , corresponding to G143A substitution related to strobilurin resistance. Specificity of the primers and amplification efficiency were preliminarily tested in conventional PCR at different annealing temperatures. The performance of several preselected primer sets was verified in real‐time PCR with SYBR Green I dye. Finally, two primer sets (‘2Wt’ and ‘5Mt’) were successfully applied for discrimination between wild‐type and mutated allele. Different sensitivity of the detection of homologous and non‐homologous DNA corresponded to the difference in Ct values equalled 10.5 for ‘2Wt’ and 12.0 for ‘5Mt’ primer set. Primers specific to Mycosphaerella fijensis cytochrome b sequence ( Wille et al. 2002 ) were applied for additional control of PCR inhibitors. The reliability of the new method was evaluated and verified using two series of reference DNA dilutions and artificial mixtures of both types of DNA. Developed real‐time PCR assay was applied to measure the ratio of mutated to non‐mutated allele in field samples, collected in Poland in 2009, using two series of reference DNA in every run. The measured mutation level for the samples derived from orchards with conventional chemical control was very high (50–100%). For two populations originating from one organic orchard, the measured level of mutation was 1% and 46%. The combination of molecular and traditional tests for the evaluation of mutation level and monitoring of resistance levels in orchards is recommended.