Molecular and biological characterization of Alternaria alternata isolates, causing ginseng leaf and stem blight, resistant to tebuconazole in Jilin Province, China

Ginseng Alternaria leaf and stem blight (GALSB), caused by Alternaria spp., is a significant above-ground disease that severely impacts both the yield and quality of ginseng. Tebuconazole has been widely used for many years as a registered fungicide to control GALSB, however, its efficacy has declined in recent years. To elucidate the resistance mechanism of A. alternata to tebuconazole, this study assessed the sensitivity of A. alternata isolates using the mycelial growth rate method, evaluated resistance levels and risk, and investigated resistance mechanisms. The results showed a baseline sensitivity of A. alternata to tebuconazole at 3.90 μg/mL, with a low resistance frequency of 11.30%, and no moderately or highly resistant isolates were detected. Three genetically stable resistant mutants were obtained through fungicide adaptation. Compared to their parental strains, these mutants exhibited reduced mycelial growth, conidial production and germination, and pathogenicity, yet showed enhanced tolerance to temperature fluctuations and osmotic stress. The resistance risk was classified as low, and the mutants showed weak competitiveness under field conditions. A G462S point mutation in the CYP51 gene was identified in the resistant strains. Molecular docking analysis confirmed that this mutation reduces the binding affinity between tebuconazole and its target protein (CYP51). Expression analysis revealed significant upregulation of CYP51 gene in the resistant mutants compared to the parental strain. In summary, A. alternata exhibits a low level and risk of resistance to tebuconazole; point mutation and overexpression of the CYP51 gene are the primary mechanisms contributing to the development of resistance. These findings provide a scientific basis for the rational use of tebuconazole in controlling GALSB. Monitoring these resistance mechanisms will facilitate the development of effective resistance management strategies, such as rotation and combined application of fungicides, to achieve sustainable and environmentally friendly disease control.