Fine Tuning of Real Time PCR as a First Tool for the Detection of G143A Substitution in Venturia inaequalis Samples

Apple scab caused by Venturia inaequalis (Cke.) Wint. is the most important disease of apple trees worldwide and requires a high number of fungicide applications. The G143A substitution in the inhibitor binding site of cytochrome b of V. inaequalis confers a high level of resistance to strobilurins targeting the bc1 complex. The aim of this work was to substitute the labor intensive in vitro assays, with the faster quantitative PCR. An allele-specific qPCR method with a newly designed primer set was successfully developed to quantitatively determine the frequency of QoI-resistant A143 allele in populations of V. inaequalis. To be able to suggest that the molecular method could be applied as unique and robust technique, we carried out in vitro sensitivity test to trifloxystrobin; first testing the relative germination and subsequently confirmed with the quantification of mutated allele frequencies by qPCR on forty-nine Italian V. inaequalis populations. qPCR gave a similar pattern to that obtained using in vitro conidial germination test in predominantly sensitive and resistant populations, the variability between these two tests was observed in some heterogeneous populations. The qPCR assay developed in this study efficiently quantifies the A143 allele and we can conclude that this method could be useful for the study of the fungicide resistance at population level in the fields, giving a quick response also with a large amount of samples.