A real-time pcr quantitative analysis of the venturia inaequalis cytb gene g143a mutation and its prevalence in the Czech republic

Abstract

Venturia inaequalis (Cooke) G. Winter (1875) is a fungal pathogen that causes a disease in apples known as apple scab, which is a major source of eco-nomic losses for apple growers. To contain the patho-gen, fungicides have been usually applied, often from the strobilurin family. Unfortunately, only two years after the introduction of strobilurin fungicides, resistant V. inaequalis populations appeared, and a substi-tution of glycine at position 143 with alanine (G143A) in the mitochondrial cytb gene was found to be the mutation responsible. Many methods exist to detect this mutation, but they are generally not suitable for simple routine use, do not provide quantitative data, or are not sensitive enough. Moreover, the quality and quantity of isolat-ed DNA from fungi may also be an issue due to the fre-quent presence of PCR inhibitors. A new real-time PCR-based assay was therefore designed that can detect 0.1% of the G143A mutation in a wild type background and vice versa; in parallel, DNA isolation was optimized, using 10 million conid-ia in a suspension prepared from leaves harvested in orchards instead of the commonly used scab mycelia. The assay was then used for screening of the resistant V. inaequalis in orchards. The G143A mutation was present in all conventional and organic orchards tested, and in the vast majority it was present at near-ly or at 100%, indicating that the mutation does not likely affect the fitness of V. inaequalis and the mutation persists over time. Furthermore, the G143A mutation (average 2.3%) was also observed in V. in-aequalis isolates harvested from alley trees along roads, implying that the mutation also occurs naturally at low levels.