Multiple mutations across the succinate dehydrogenase gene complex are associated with boscalid resistance in Didymella tanaceti in pyrethrum

Failures in control of tan spot of pyrethrum, caused by Didymella tanaceti, has been associated with decreased sensitivity within the pathogen population to the succinate dehydrogenase inhibitor (SDHI) fungicide boscalid. Sequencing the SdhB, SdhC, and SdhD subunits of isolates with resistant and sensitive phenotypes identified 15 mutations, resulting in three amino acid substitutions in the SdhB (H277Y/R, I279V), six in the SdhC (S73P, G79R, H134R, H134Q, S135R and combined H134Q/S135R), and two in the SdhD (D112E, H122R). In vitro testing of their boscalid response and estimation of resistance factors (RF) identified isolates with wild-type (WT) Sdh genotypes were sensitive to boscalid. Isolates with SdhB-I279V, SdhC-H134Q and SdhD-D112E exhibited moderate resistance phenotypes (10 ≥ RF < 100) and isolates with SdhC-H134R exhibited very high resistance phenotypes (RF ≥ 1000). All other substitutions were associated with high resistance phenotypes (100 ≥ RF < 1000). High-resolution melt assays were designed and used to estimate the frequencies of substitutions in four field populations (n = 774) collected in August (pre-boscalid application) and November (post-boscalid application) 2012. The SdhB-H277Y, SdhC-H134R and SdhB-H277R genotypes were most frequently observed across populations at 56.7, 19.0, and 10.3%, respectively. In August 92.9% of D. tanaceti contained a substitution associated with decreased sensitivity. Following boscalid application, this increased to 98.9%, with no WT isolates detected in three fields. Overlaying previously obtained microsatellite and mating-type data revealed that all ten recurrent substitutions were associated with multiple genotypes. Thus, boscalid insensitivity in D. tanaceti appears widespread and not associated with clonal spread of a limited pool of individuals.