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

Abstract

Fourteen primers were designed specific to SNP (G fi 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 (Ô2WtÕ and Ô5MtÕ) 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 Ô2WtÕ and 12.0 for Ô5MtÕ 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.

Use of QuantitativePCRDetection Methods to Study Biocontrol Agents and Phytopathogenic Fungi and Oomycetes in Environmental Samples

“…Similarly, a SNP in the cytochrome b gene of Venturia inaequalis , corresponding to G143A substitution related to strobilurin resistance, was monitored by qPCR and revealed a higher mutation level in populations from conventional chemical control fields as compared to an organic orchard (Michalecka et al. ).…”

Efficacy loss of strobilurins used in protection against apple scab in Czech orchards

“…The speed of mutants' propagation depends on the pathogen biology as well as the number of fungicide applications per season (Sierotski et al 2000;Barr et al 2005;Lesemann et al 2006;Chen et al 2007). Last research objectives lead to developing of different detection techniques for monitoring of causal mutations (Michalecka et al 2011).…”

Identification of Neofabraea species causing bull's eye rot of apple in Poland and their direct detection in apple fruit using multiplex PCR

“…DNA extraction from apple fruit skin of all 550 samples collected from apple fruits with disease symptoms and 120 samples of symptomless apple fruits, was conducted according to the method of Doyle & Doyle () with modifications described by Michalecka et al . (). Of the DNA samples obtained, 17% needed to be purified further; this step was performed using Axyprep Multisource Genomic DNA Miniprep kit.…”