Evidence of strobilurine resistant isolates of A. solani and A. alternata in Germany

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1 Technische Universität München Evidence of strobilurine resistant isolates of A. solani and A. alternata in Germany H. Hausladen, B. Adolf and J. Leiminger

2 outline introduction material and methods results Alternaria solani Alternaria alternata summary and discussion

3 EB control with strobilurines EB control by strobilurines widely used due to important benefits and excellent disease control mode of action is highly specific (single site mode of action) high risk of fungicide resistance first evidence of the F129L mutation in A. solani by Gudmestad (2000) and G143A in A. alternata by Ma et al. (2003)

4 resistance to QoI fungicides effects Alternaria solani: F129L mutation resulting in a partial resistance (Gisi, Sierotzki, 2008) reduced fungicide sensitivity, reduced disease control (see also in Pyrenophora teres) Alternaria alternata: G143A mutation resulting in a high level of resistance (Gisi, Sierotzki, 2008) complete loss of disease control by use of QoI fungicides (see also in Mycosphaerella graminicola)

5 isolate monitoring examination of leaf samples for their occurence of Alternaria species (A. solani, A. alternata) German wide monitoring period:

6 monitoring 2008

7 monitoring 2010

8 detection of the F129L mutation in A. solani Technische Universität München

9 detection of the F129L mutation in A. solani Technische Universität München site mutation at position 129 from TTC to TTA, CTC, TTG substitution from phenylalanin to leucin (Phe Leu) F 129 L F129 Phenylanalin Leucin

10 detection of two different genotypes within in A. solani Technische Universität München Alt. sol. type II - USA 207 bp AGAA... exon ATGGCTACAGCTTTCCTGGGT Intron (139 bp sequenced, 90% homology to intron bi3 of P.teres (Sierotzky et al. 2007)... A126 G131 Alt. sol. type I - Europe 214 bp exon Intron (1140 bp) exon Intron (2157 bp) exon Intron (1760 bp) AGAA... ATGGCT... ACAGCTTTCCTGGGTTATGTTCTTCCTTATGGGCAAATGTCTTTATGAGGT A126 G131 G GCTACAGTT... (Grasso et al. 2006)

11 detection of the F129L mutation in A. solani Technische Universität München site mutation at position 129 from TTC to TTA, CTC, TTG substitution from phenylalaninen to leucin (Phe Leu) sequenzing of a 207 and 214 bp fragment of the cyt b complex in A. solani F 129 L F129 Phenylanalin Leucin

12 isolate-monitoring year isolates nr of locations Strobilurin applied Locations with F129L Nr mutants Frequency % F129L 5,1 15,3 56,5 17,0 37,7 32,2

13 detection of two different genotypes within in A. solani Technische Universität München Alt. sol. type II - USA 207 bp AGAA... exon ATGGCTACAGCTTTCCTGGGT Intron (139 bp sequenced, 90% homology to intron bi3 of P.teres (Sierotzky et al. 2007)... A126 G out of 147 F129L mutants were genotype II, carrying TTA, TTG mutation Alt. sol. type I - Europe 214 bp exon Intron (1140 bp) exon Intron (2157 bp) exon Intron (1760 bp) AGAA... ATGGCT... ACAGCTTTCCTGGGTTATGTTCTTCCTTATGGGCAAATGTCTTTATGAGGT A126 G131 G GCTACAGTT... (Grasso et al. 2006) only 3 out of 147 F129L mutants, were genotype I, carrying CTC mutation first observed in 2013 (2 isolates) and 2014 (one isolate) all wildtype isolates were genotype I

14 appearance of isolates with F129L mutation Wild-type isolate F129L mutant

15 site-specific distribution of F129L and wild-type isolates Technische Universität München investigation of various isolates out of each sampled location year sampling date aug 16 th aug 16 th aug 16 th sep 11 th aug 28 th sep 18 th sep 18 th location Aschheim Aiterhofen Kirchheim Laberweinting Freising Freising Freising treatment AZ, 4 times AZ, 4 times AZ, 4 times AZ, 3 times unknown untreated AZ, 4 times number of isolates proportion of wild-type isolates proportion of F129L isolates constant site-specific composition of species

16 site-specific occurence of F129L mutants Technische Universität München A. solani location with F129L mutant wild-type wild-type and F129L mutants

17 fungicide sensitivity tests (EC 50 -value) evaluation of fungicide sensitivity of A. solani wild-type and F129L mutants EC 50, US reference F129L mutants EC 50, US reference WT strains EC 50: concentration, at which 50 % of the spores die or do not germinate

18 fungicide sensitivity tests (EC 50 -value) evaluation of fungicide sensitivity of A. solani wild-type and F129L mutants EC 50, US reference F129L mutants EC 50, US reference WT strains

19 fungicide sensitivity tests (EC 50 -value) evaluation of fungicide sensitivity of A. solani wild-type and F129L mutants 10,2 EC 50, US reference F129L mutants EC 50, US reference WT strains

20 detection of the G143A mutation in A. alternata Technische Universität München

21 detection of the G143A mutation in A. alternata Technische Universität München Point mutation within the cyt b complex, mutation site 143 GGT -> GCT (Gly Ala) G 143 A analysis of 150 single spore isolates ( ) and 58 EB infected leaf samples Sequencing of an 228 bp fragment cyt b incl. G143A mutation Primer: AF (5-ACA CTG CTT CAG CAT TTT TCT TCA TAG-3) AR (5-TTG TCC AAT TCA TGG TAT AGC ACT CA-3) (Ma et al. 2003) A.alt GATACGTCTTGCCATACGGGCAAATGTCATTATGAGCTGCAACAGTT A.alt GATACGTCTTGCCATACGGGCAAATGTCATTATGAGCTGCAACAGTT A.alt 11 - GATACGTCTTGCCATACGGGCAAATGTCATTATGAGGTGCAACAGTT A.alt 12 - GATACGTCTTGCCATACGGGCAAATGTCATTATGAGGTGCAACAGTT M M WT WT

22 investigated isolates in total isolates evaluated 7 43 / / wildtyp isolates 7 34 / / G143A mutants 0 9 / / investigated locations 1 29 / / locations with G143A 0 9 / /

23 Site-specific occurence of G143A mutations Technische Universität München A. alternata location with G143A mutant wild-type wild-type and G143A mutants

24 Fungicide sensitivity tests (EC 50 -value) with azoxystrobin Technische Universität München evaluation of fungicide sensitivity of A. alternata wild-type and G143A mutants wild-type isolates Germination % Germination % G143A isolates

25 Summary prevalence of the F129L and G143A mutation in Germany actually no loss in fungicide sensitivity (field) further specific field trials in 2015 (artificial inoculation) use of QoI fungicides to GAP: limitation of Strobilurin-containing products Use products/mixtures with high efficacy Full dosage Use of EB fungicides first spray: 7-8 weeks after the crop emerge further applications: according to disease development and weather condition

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