Resistance Development to Procymidone and Boscalid in Alternaria alternata Causing Black Spot Disease on Fritillaria thunbergii

Fritillaria thunbergii is a valuable medicinal plant threatened by black spot disease caused by Alternaria alternata for which application of fungicides is the primary strategy. However, the detection and characterization of fungicide resistance status in F. thunbergii has not yet been reported. A total of 106 and 128 single-spore isolates of A. alternata were recovered in 2015 and 2021, respectively, from 52 and 56 F. thunbergii fields located in Jinhua, Wenzhou, Hangzhou, and Ningbo, where 90% of F. thunbergii is produced. In 2015, moderate resistance to procymidone (Pro MR ), a dicarboximide fungicide that has been applied for more than 10 years, was detected at a rate of 23.6%, and the resistance frequency increased to as high as 44.5% in 2021. For the SDHI boscalid, the EC 50 values ranged from 0.013 to 0.976 μg/ml, with the baseline EC 50 value of 0.57 ± 0.29 μg/ml in 2015, while the sensitivity profile showed prominent characteristics of a disruptive distribution, with a resistance frequency of 22.6% in 2022. Positive cross-resistance was observed between procymidone and iprodione (ρ = 0.897, P = 0.000) and between boscalid and pydiflumetofen (ρ = 0.937, P = 0.000). No fitness penalty in growth, sporulation, germination, or pathogenicity was found for the Pro MR or the boscalid-resistant (Bos R ) isolates. P894L of Os1 was observed in all the tested Pro MR isolates, and the double point mutation (P894L + S1277L) was detected in 33.3% of the Pro MR isolates. Four types of mutations in Sdh (G14W in Sdh B, V181G in Sdh D, A47T in Sdh D, and G14W in Sdh B + A47T in Sdh D) were found in the Bos R isolates.