First Report and Characterization of Propiconazole Resistance in <i>Tilletia horrida</i> Causing Kernel Smut in Rice

Rice kernel smut, caused by Tilletia horrida, poses a major threat to rice production in the US. To manage this disease, growers primarily rely on the midseason preventive applications of propiconazole, a fungicide. Propiconazole belongs to the class of demethylation inhibitors (DMIs) that, hinders the fungal sterol synthesis. In recent years, reports of reduced efficacy and failures of propiconazole fungicides in managing kernel smut have become increasingly widespread across the US. This study aimed to assess the resistance of T. horrida isolates to propiconazole and to identify the molecular basis of the resistance. In vitro tests were conducted using three T. horrida isolates from organic rice fields with no history of fungicide applications to establish a baseline EC50 for propiconazole, which was determined to be 0.02 mg/L. We screened for resistance with additional 66 T. horrida isolates collected from various regions across the US. The results revealed that 84% of the isolates exhibited EC50 exceeding the baseline of 0.02 mg/L. Among these, 57% had EC50 values above 0.05 mg/L, while 39% and 23% showed EC50 greater than 1 mg/L and 2 mg/L, respectively. Further genome analysis of the T. horrida unveiled the presence of a single copy of the Cyp51 gene, the target of DMIs. The sequence analysis of the Cyp51 protein in propiconazole-resistant T. horrida isolates revealed five amino acid substitutions: G22A, R183K, V279A, L387I, and G494S. This study marks the first investigation into propiconazole resistance in T. horrida and its association with amino acid mutations in the Cyp51 gene. These findings highlight an urgent need to search for alternative fungicides with different modes of action to effectively manage kernel smut in rice.