Characteristics of fungicide resistance in Asian soybean rust ( Phakopsora pachyrhizi ) in Paraguay for 2022–2023: insights from genetic mutations and molecular dynamics simulations

BACKGROUND Phakopsora pachyrhizi , the causative agent of Asian soybean rust, poses a major threat to soybean production in South America. The rising incidence of fungicide resistance underscores the need for a deeper understanding of its underlying genetic mechanisms. This study characterized fungicide resistance profiles in P. pachyrhizi from Paraguay, integrating genetic mutation analyses with molecular dynamics simulations to elucidate resistance mechanisms and inform disease management strategies. RESULTS The first characterization of fungicide resistance profiles in P. pachyrhizi from eastern Paraguay (2022–2023) revealed multiple resistance patterns to demethylation inhibitor (DMI), succinate dehydrogenase inhibitor (SDHI), and quinone outside inhibitor (QoI) fungicides. Detached leaf assays demonstrated reduced fungicidal efficacy relative to susceptible control strains. Pyrosequencing revealed mutations in CYP51 (F120L, V130A, Y131H/F, I145V), SDH‐C (I86F), and CYTB (F129L), with variable mutation values across populations and isolates. Notably, isolates from a single field displayed substantial genetic and phenotypic variability, indicating complex intra‐field resistance dynamics. CYP51 mutations were markedly overexpressed in cDNA relative to gDNA, reinforcing their contribution to DMI resistance. Molecular dynamics simulations of prothioconazole‐desthio interactions revealed key structural determinants of resistance, in which protein‐water‐ligand networks played a critical role in modulating fungicide efficacy. CONCLUSION These findings provide critical insights into the genetic basis of fungicide resistance in P. pachyrhizi and highlight the necessity for continuous molecular surveillance, resistance management, and strategic adaptation of fungicide applications to mitigate the spread of resistant strains in soybean‐growing regions of South America.