Universität Hohenheim Institut für Phytomedizin Fachgebiet Phytopathologie Prof. Dr. Ralf T. Vögele Analysis of the emerging situation of resistance to succinate dehydrogenase inhibitors in Pyrenophora teres and Zymoseptoria tritici in Europe Dissertation zur Erlangung des Grades eines Doktors der Agrarwissenschaften vorgelegt der Fakultät Agrarwissenschaften von Dipl. Agr. Biol. Alexandra Rehfus aus Leonberg 2018 Diese Arbeit wurde unterstützt und finanziert durch die BASF SE, Unternehmensbereich Pflanzenschutz, Forschung Fungizide, Limburgerhof. Die vorliegende Arbeit wurde am 15.05.2017 von der Fakultät Agrarwissenschaften der Universität Hohenheim als „Erlangung des Doktorgrades an der agrarwissenschaftlichen Fakultät der Universität Hohenheim in Stuttgart“ angenommen. Tag der mündlichen Prüfung: 14.11.2017 1. Dekan: Prof. Dr. R. T. Vögele Berichterstatter, 1. Prüfer: Prof. Dr. R. T. Vögele Mitberichterstatter, 2. Prüfer: Prof. Dr. O. Spring 3. Prüfer: Prof. Dr. Dr. C. P. W. Zebitz Leiter des Kolloquiums: Prof. Dr. J. Wünsche Table of contents III Table of contents Table of contents ................................................................................................. III Abbreviations ..................................................................................................... VII Figures ................................................................................................................. IX Tables .................................................................................................................. XII Abstract ................................................................................................................. 1 1 Introduction .................................................................................................... 4 1.1 Wheat and barley cultivation and the importance of fungal diseases ......................... 4 1.1.1 Net blotch disease in barley caused by Pyrenophora teres ......................................................... 5 1.1.1.1 Taxonomy and relationship to other Pyrenophora species ................................................. 6 1.1.1.2 Life cycle ............................................................................................................................ 7 1.1.1.3 Infection process and toxin production ............................................................................... 8 1.1.1.4 Population diversity ............................................................................................................ 9 1.1.2 Septoria tritici blotch in wheat caused by Zymoseptoria tritici ................................................. 10 1.1.2.1 Importance ........................................................................................................................ 10 1.1.2.2 Taxonomy ......................................................................................................................... 10 1.1.2.3 Life cycle .......................................................................................................................... 10 1.1.2.4 Infection process ............................................................................................................... 12 1.1.2.5 Genetics and population diversity ..................................................................................... 13 1.2 Disease management in cereals ..................................................................................... 13 1.2.1 Cultural measures ...................................................................................................................... 14 1.2.2 Resistance breeding ................................................................................................................... 14 1.2.3 Chemical control using fungicides ............................................................................................ 15 1.2.4 Succinate dehydrogenase inhibitors (SDHIs) ............................................................................ 18 1.2.5 Differences in fungicide use in European countries .................................................................. 21 1.3 Evolution of fungicide resistance .................................................................................. 22 1.3.1 Resistance to succinate dehydrogenase inhibitors ..................................................................... 25 1.3.2 Fungicide resistance in P. teres and Z. tritici............................................................................. 26 2 Objectives ..................................................................................................... 28 3 Material and Methods .................................................................................. 29 3.1 Technical devices ............................................................................................................ 29 3.2 Chemicals and consumables .......................................................................................... 30 Table of contents IV 3.3 Enzymes, kits and bacterial strain ................................................................................ 31 3.4 Buffer und solutions ....................................................................................................... 32 3.5 Growth media ................................................................................................................. 33 3.6 Oligonucleotides ............................................................................................................. 34 3.7 Software .......................................................................................................................... 37 3.8 Cultivation of living organisms ..................................................................................... 37 3.8.1 Cultivation of wheat and barley plants ...................................................................................... 37 3.8.2 Cultivation of fungal isolates ..................................................................................................... 37 3.9 Fungal isolates and leaf samples ................................................................................... 38 3.9.1 ‘Random monitoring’ and ‘Trial site monitoring’ ..................................................................... 38 3.9.2 ‘Field monitoring’...................................................................................................................... 39 3.9.2.1 Generation of P. teres and Z. tritici from infected leaves ................................................. 40 3.10 Molecular biological methods........................................................................................ 40 3.10.1 Standard molecular techniques .............................................................................................. 40 3.10.2 Sequencing ............................................................................................................................ 42 3.10.3 SNP detection systems .......................................................................................................... 42 3.10.3.1 Quantitative real-time PCR (qPCR) .................................................................................. 43 3.10.3.2 Pyrosequencing ................................................................................................................. 44 3.11 Fungicide sensitivity tests .............................................................................................. 46 3.11.1 Fungicides used ..................................................................................................................... 46 3.11.2 Microtiter tests ...................................................................................................................... 49 3.11.3 Sensitivity tests in the glasshouse ......................................................................................... 50 3.11.3.1 Inoculation ........................................................................................................................ 50 3.11.3.2 Fungicide application ........................................................................................................ 51 3.11.3.3 Rating ................................................................................................................................ 52 3.12 Competition studies of SDHI resistant isolates ............................................................ 52 3.12.1 Competition studies in planta ............................................................................................... 52 3.12.2 Generation of SDHI-resistant mutants of Z. tritici ................................................................ 53 3.13 Studies to detect enhanced efflux of Z. tritici ............................................................... 54 3.14 Homology modelling ...................................................................................................... 55 3.15 Bioinformatic analyses ................................................................................................... 55 4 Results .......................................................................................................... 56 4.1 Emergence of SDHI resistance in Pyrenophora teres in Europe ................................ 56 4.1.1 Detection and analysis of resistant isolates ................................................................................ 56 4.1.2