CYP51 alterations in Alternaria solani and their effects on DMI sensitivity and fitness

ABSTRACT Alternaria solani, the causal agent of early blight, may dramatically reduce yield in potato production on a global scale. Quinone outside inhibitors (QoIs), succinate dehydrogenase inhibitors (SDHIs), and demethylation inhibitors (DMIs) represent three major fungicide groups that are used to control early blight on potato. Early blight has acquired adaptation to QoIs and SDHIs, making DMI fungicides the prime class of chemistries in early blight control. We investigated the current DMI sensitivity status of field isolates of A. solani . The combination of two mutations leading to the amino acid exchanges L143F and G446S was detected in the DMI target gene CYP51 in some isolates. Both mutations occurred only in combination and were linked to reduced DMI sensitivity. To test for the role of these mutations in DMI adaptation, we generated strains with single L143F or G446S mutations, as well as L143F + G446S double mutations by targeted mutagenesis. Single mutations caused slightly lower DMI sensitivity; this was more pronounced with the mutation combination L143F + G446S. Importantly, in vivo infection assays performed in the greenhouse revealed significantly lower competitiveness of the double mutants compared to wild-type isolates, indicating that the L143F + G446S haplotype is connected with lower fitness. However, CYP51 haplotypes do not differ in spore morphology or quantity. Furthermore, CYP51 haplotypes generated by targeted mutagenesis are all able to infect tomato plants in the greenhouse. IMPORTANCE This study investigates the mutations in the CYP51 gene of the fungus Alternaria solani , which causes early blight. By understanding the effect of these mutations, researchers can better manage fungicide resistance, ensuring effective disease control. This study provides biotechnological methods suitable for investigating single-site mutations and their individual effect on fungicide sensitivity.