title: A pyrosequencing-based method to quantify genetic substitutions associated with resistance to succinate dehydrogenase inhibitor fungicides in Botrytis spp. populations.
abstract: BACKGROUND
  The genetic underlying resistance mechanisms in the population of the phytopathogenic fungus Botrytis cinerea are well documented. Specifically, several genetic substitutions associated with succinate dehydrogenase inhibitor (SDHI)-based fungicide resistance have been identified in the succinate dehydrogenase gene. The objective of the present work was to develop a molecular tool for accurate quantification of these genetic substitutions within Botrytis populations. A test using the PyroMark Q24 instrument was designed to detect and quantify five genetic substitutions associated with SDHI resistance.
  
  
  RESULTS
  The technique is based on sequencing by synthesis, and it generated quantitative and accurate data with a limit of quantification of a minimum of 500 spores. There was a linear relationship between the known and estimated percentages of spores with the targeted genetic substitutions and wild-type strains at ratios of 0-100%, with a 20% increment.
  
  
  CONCLUSIONS
  With the pyrosequencing assay developed in this study, a large number of Botrytis spp. individuals can be characterised in a timely fashion with greater accuracy than by commonly used methods. Hence, pyrosequencing-based methods will be useful for improving our understanding of fungicide resistance, detecting the arrival of new genetic substitutions, monitoring shifts in fungal populations and assessing the effectiveness of antiresistance strategies, and for routine monitoring of fungicide resistance.
authors.affiliation: Biology Department, University of Sherbrooke, Sherbrooke, Quebec, Canada.|Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Gobeil-Richard
authors.affiliation: Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada.
authors.forename: David-Mathieu
authors.initials: DM
authors.lastname: Tremblay
authors.affiliation: Biology Department, University of Sherbrooke, Sherbrooke, Quebec, Canada.
authors.forename: Carole
authors.initials: C
authors.lastname: Beaulieu
authors.affiliation: Compagnie de Recherche Phytodata Inc., Sherrington, Quebec, Canada.
authors.forename: Hervé
authors.initials: H
authors.lastname: Van der Heyden
authors.affiliation: Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada.
authors.forename: Odile
authors.initials: O
authors.lastname: Carisse
mesh_terms: D020171:Botrytis / Q000187:drug effects* / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics*; D005659:Fungicides, Industrial / Q000494:pharmacology*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013385:Succinate Dehydrogenase / Q000037:antagonists & inhibitors*
chemical_list: D005659:Fungicides, Industrial; D013385:Succinate Dehydrogenase
keywords: Botrytis; PyroMark Q24; SDHI; fungicide resistance; pyrosequencing