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

<i>Fritillaria thunbergii</i> is a valuable medicinal plant threatened by black spot disease caused by <i>Alternaria alternata</i> for which application of fungicides is the primary strategy. However, the detection and characterization of fungicide resistance status in <i>F. thunbergii</i> has not yet been reported. A total of 106 and 128 single-spore isolates of <i>A. alternata</i> were recovered in 2015 and 2021, respectively, from 52 and 56 <i>F. thunbergii</i> fields located in Jinhua, Wenzhou, Hangzhou, and Ningbo, where 90% of <i>F. thunbergii</i> 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 EC₅₀ values ranged from 0.013 to 0.976 μg/ml, with the baseline EC₅₀ 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, <i>P</i> = 0.000) and between boscalid and pydiflumetofen (<i>ρ</i> = 0.937, <i>P</i> = 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 <i>O</i>s<i>1</i> 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 <i>Sdh</i> (G14W in <i>Sdh</i> B, V181G in <i>Sdh</i> D, A47T in <i>Sdh</i> D, and G14W in <i>Sdh</i> B + A47T in <i>Sdh</i> D) were found in the Bos^R isolates.