Naturally occurring target‐site EPSPS mutations and moderate EPSPS over‐expression confer glyphosate resistance in Lolium perenne ssp. multiflorum biotypes from Argentina

BACKGROUND: Glyphosate resistance in Lolium perenne ssp. multiflorum (Italian ryegrass), a major weed in temperate cropping systems, threatens effective weed control and crop productivity. This study characterized the glyphosate response and target-site glyphosate resistance mechanisms in three biotypes (PA, MG and HE) from northwest Pampas region. RESULTS: ) values, with resistance indices ranging from 5.5 to 9.3. Both resistant biotypes (MG and HE) displayed hormesis, showing growth stimulation at sublethal glyphosate doses. Shikimic acid accumulation assays confirmed reduced glyphosate sensitivity in resistant biotypes, consistent with lower levels of EPSPS inhibition. Partial EPSPS gene sequencing revealed distinct Pro-106 substitutions: Pro-106-Ser in HE, and Pro-106-Thr in PA and MG, both previously associated with reduced glyphosate binding while maintaining enzyme activity. These results suggested at least two independent evolutionary origins of this resistance. Quantitative PCR revealed significantly increased EPSPS transcript levels in resistant biotypes without evidence of gene copy number amplification, indicating that elevated EPSPS expression may contribute to resistance in combination with target-site mutations. CONCLUSION: Glyphosate resistance in Italian ryegrass is primarily associated with target-site EPSPS mutations (Pro-106-Ser and Pro-106-Thr), whereas increased EPSPS expression may further contribute to the resistant phenotype. Hormesis in some resistant biotypes adds complexity to the resistance dynamics. Together, these findings highlight the adaptive potential of this species under recurrent glyphosate selection and underscore the need for integrated weed management strategies to delay the evolution and spread of herbicide resistance.