First Report of QoI Resistance in Venturia inaequalis Causing Apple Scab in Apple Orchards in Turkey

Apple scab caused by Venturia inaequalis (Cke.) Wint. is the most important disease of apple (Malus × domestica) in Turkey. To control apple scab, quinone outside inhibitor (QoI) fungicides have been used since 1998. Apple scab control failures were observed in 2005 in Mediterranean and Central Anatolian regions, where apple production is intensive and where QoIs have been applied in 3 or 4 consecutive applications per season from pink bud stage to half fruit size of apple trees. Ten field populations of V. inaequalis, each obtained from 40 to 50 infected leaves, were collected in orchards located in the aforementioned production regions in May 2011 to verify the sensitivity to QoI fungicides. Samples were obtained from one wild-type orchard which had never been treated with fungicides and was far away from the treated orchards, four orchards which had never been exposed to QoIs, and five orchards where QoIs control failures were observed. To obtain populations with high germinative energy, conidia were propagated on apple seedlings (cv. Golden) in greenhouse (Küng Färber et al. 2002). After 20 days of incubation, conidial suspension (1 to 3 × 105 spores/ml) was prepared from infected V. inaequlis leaves for the evaluation of spore germination on agar media amended with 0, 0.001, 0.01, 0.1, and 2 mg/liter of trifloxystrobin as technical grade (Sigma-Aldrich, St. Louis, MO) (Fiaccadori et al. 2011). Data were transformed into the 50% effective concentration (EC50) by probit analysis utilizing Microsoft Excel. Genomic DNA was extracted directly from lyophilised infected leaves ground in liquid nitrogen by a CTAB-based method and then purified through a Sepharose 6B (Bramwell et al. 1995). CAPS analyses were performed with two specific primers, ANK 10 and ANK 283, to amplify a 413-bp fragment of cytochrome b gene which includes the G143 codon following the protocol by Fiaccadori et al. 2011. qPCR was performed for the quantitative detection of the A143 allele using an ICycler-IQ5 (Bio-Rad) with SybrGreen (Bio-Rad) with the primer set both for the wild-type (FwS5418-Rv5548) and for the mutant allele (FwR5418-Rv5548) (Nanni et al. 2011). The ratio (%) of the two alleles present in samples was calculated using the ΔCq method according to the equation: 10^ (Cq S allele– Cq R allele)/-slope); R (R/1+R) × 100 = R allele. Data are expressed as a percentage of the mutated allele. The wild-type population had an EC50 value of 0.00001 mg/liter and the four untreated orchards showed EC50 values ranging from 0.004 to 0.04 mg/liter. These V. inaequalis orchard populations can be considered sensitive according to the baseline study (Küng Färber et al. 2002). qPCR detected R-allele frequencies ranging from 0.1 to 3.61% in these V. inaequalis populations, whereas G143A substitution was not detected by CAPS PCR because its detection limit is under 10% (Bäumler et al. 2003). The five populations obtained from orchards where poor control has been reported were defined as resistant, showing EC50 values ranging from 1.46 to >2 mg/liter. These populations exhibited the G143A substitution with a higher R-allele frequency, compared with the wild-type population, ranging from 46.62 to 99.76%. Consequently, practical resistance of V. inaequalis to QoI is present in some Turkish apple-growing areas. To our knowledge, this is the first report of practical QoI fungicide resistance and G143A detection in V. inaequalis in Turkey that led to limiting QoI use to one application per season in the sampled areas.