Citation: Heaven, T.; Armitage, A.D.; Xu, X.; Goddard, M.R.; Cockerton, H.M. Dose-Dependent Genetic Resistance to Azole Fungicides Found in the Apple Scab Pathogen. J. Fungi 2023, 9, 1136. https://doi.org/ 10.3390/jof9121136 Academic Editors: Zonghua Wang and Jun Huang Received: 14 September 2023 Revised: 13 November 2023 Accepted: 17 November 2023 Published: 24 November 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Fungi Journal of Article Dose-Dependent Genetic Resistance to Azole Fungicides Found in the Apple Scab Pathogen Thomas Heaven 1,2,3, *, Andrew D. Armitage 4 , Xiangming Xu 1 , Matthew R. Goddard 2 and Helen M. Cockerton 5 1 National Institute of Agricultural Botany, New Road, East Malling, West Malling, Kent ME19 6BJ, UK; xiangming.xu@niab.com 2 The School of Life and Environmental Sciences, University of Lincoln, Lincoln LN6 7DL, UK; mgoddard@lincoln.ac.uk 3 John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK 4 Natural Resources Institute, University of Greenwich, Kent ME4 4TB, UK; a.d.armitage@greenwich.ac.uk 5 School of Biosciences, University of Kent, Canterbury CT2 7NZ, UK; h.cockerton@kent.ac.uk * Correspondence: thomas.heaven@jic.ac.uk; Tel.: +44-1603450060 Abstract: The evolution of azole resistance in fungal pathogens presents a major challenge in both crop production and human health. Apple orchards across the world are faced with the emergence of azole fungicide resistance in the apple scab pathogen Venturia inaequalis. Target site point mutations observed in this fungus to date cannot fully explain the reduction in sensitivity to azole fungicides. Here, polygenic resistance to tebuconazole was studied across a population of V. inaequalis. Genotyp- ing by sequencing allowed Quantitative Trait Loci (QTLs) mapping to identify the genetic components controlling this fungicide resistance. Dose-dependent genetic resistance was identified, with distinct genetic components contributing to fungicide resistance at different exposure levels. A QTL within linkage group seven explained 65% of the variation in the effective dose required to reduce growth by 50% (ED 50 ). This locus was also involved in resistance at lower fungicide doses (ED 10 ). A second QTL in linkage group one was associated with dose-dependent resistance, explaining 34% of variation at low fungicide doses (ED 10 ), but did not contribute to resistance at higher doses (ED 50 and ED 90 ). Within QTL regions, non-synonymous mutations were observed in several ATP-Binding Cassette and Major Facilitator SuperFamily transporter genes. These findings provide insight into the mechanisms of fungicide resistance that have evolved in horticultural pathogens. Identification of resistance gene candidates supports the development of molecular diagnostics to inform management practices. Keywords: quantitative trait loci (QTLs); plant pathogen; biotroph; linkage map; genotyping by sequencing; single nucleotide polymorphism (SNP); genome; dose response; polygenic; tebuconazole 1. Introduction Apple scab, caused by the fungal plant pathogen Venturia inaequalis, is one of the most damaging diseases impacting apple production [1–3]. Infections via airborne spores result in characteristic “scab” like lesions on both leaves and fruit; these lesions render fruits unacceptable for direct-to-consumer retail. Extensive control measures are, therefore, required to prevent scab outbreaks and mitigate the severe economic impact of the disease. It is estimated that apple producers in the eastern United States alone spend in excess of USD 18.6 million per year on scab control [4]. Many fungicides are registered for the control of apple scab, and growers are advised to utilise products with differing modes of action to prevent the development of fungicide resis- tance [5]. Despite this, V. inaequalis has evolved resistance to successive classes of fungicides going back to the 1950s [6]. This resistance has often developed quickly after the introduction of a new active and been maintained in the pathogen population thereafter [6]. Combined J. Fungi 2023, 9, 1136. https://doi.org/10.3390/jof9121136 https://www.mdpi.com/journal/jof