Photosystem II-encoded terbuthylazine resistance in Serbian Solanum nigrum L.: evidence for a novel psbA N267K mutation coupled with enhanced cytochrome P450 activity

The intensive application of triazine herbicides, particularly terbuthylazine, has led to the evolution of PSII-inhibitor resistance in Solanum nigrum species in Serbian maize fields. The present study was conducted to identify the mechanisms conferring resistance to terbuthylazine herbicides in a S. nigrum biotype and to explore effective chemical alternatives for managing this resistance. Dose-response studies revealed that the resistant biotype is 12 times more resistant to terbuthylazine than the susceptible biotype. In contrast, herbicides with alternative modes of action remained highly effective, indicating viable management options. However, bentazone exhibited lower efficacy than the other active ingredients tested. Prior treatment with malathion, a known inhibitor of cytochrome P450s, reduced the level of resistance to terbuthylazine in the resistant biotype. Chlorophyll fluorescence measurement revealed that the R biotype maintained an Fv/Fm value close to that of the untreated control across all doses and time points. In contrast, the S biotype exhibited a steady, dose-dependent decline, with a decrease in Fv/Fm of approximately 10–15% at the field rate compared to the untreated control. No previously known mutations were detected; however, our study identified three novel mutations, including heterozygous substitutions Ala250Pro (A250P) and Asp267Lys (N267K), and one homozygous mutation, Gly256Val (G256V). Molecular docking and dynamics analyses demonstrated that among these substitutions, N267K had the strongest functional impact. This mutation disrupted terbuthylazine binding within the PSII binding pocket by eliminating key hydrogen bonds and electrostatic interactions, thereby reducing herbicide affinity. Novel target gene mutation at N267K and enhanced metabolism by cytochrome P450s are likely mechanisms of resistance to terbuthylazine in S. nigrum. This study also established chlorophyll fluorescence measurement as a rapid and reliable method for confirming herbicide resistance in this weed species. The current findings underscore the need to avoid the use of PSII-inhibiting herbicides, particularly triazines, to prevent further increasing selection pressure. Instead, they can use alternative modes of action of herbicides for better efficacy and to reduce the risk of herbicide resistance.