The I4746K mutation in the ryanodine receptor is associated with high‐level resistance to diamide insecticides in Phthorimaea absoluta

BACKGROUND: The South American tomato pinworm, Phthorimaea absoluta, has developed resistance to diamide insecticides mainly through target-site mutations in the ryanodine receptor (RyR), including I4746M, G4903E, and G4903V. Recently, a novel mutation, I4746K, has been identified in field populations of P. absoluta, but its contribution to resistance remains unclear. RESULTS: In this study, a field population (TR-Krş-21) harboring the I4746K mutation exhibited 116.65-fold resistance to chlorantraniliprole (CHL). After six rounds of laboratory selection with CHL, resistance increased to 1109.94-fold (CHL-Sel), with a realized heritability of 0.58. Resistance remained stable for 1 year without insecticide exposure, and genetic analyses indicated that it is autosomal, incompletely dominant, and polygenic. Cross-resistance was observed among diamide insecticides, but not to abamectin or broflanilide. Synergist bioassays with piperonyl butoxide and diethyl maleate showed that inhibition of detoxification enzymes did not fully restore susceptibility. CHL selection resulted in a positive correlation between I4746K allele frequency and resistance, and genetic linkage analysis confirmed a significant association between the mutation and the resistant phenotype. Modeling and molecular dynamics simulations revealed that the I4746K substitution reshapes ligand-receptor interactions without markedly reducing overall binding affinity. Finally, a quantitative sequencing protocol and a tetra-primer amplification-refractory mutation system polymerase chain reaction assay were developed for rapid detection of the I4746K mutation. CONCLUSION: This study emphasizes the significance of the I4746K mutation in diamide resistance and offers valuable insights into how this mutation variably influences the binding of diamide insecticides, thereby informing strategies for resistance management in P. absoluta. © 2026 Society of Chemical Industry.