Temporal assessment of N-cycling genes and nutrient-acquiring enzymes under rotational grazing

Despite the importance of microbial activity for the sustainability of grazing systems, longitudinal assessments of microbial responses to grazing remain scarce. We monitored functional N-cycling genes and C-, N-, and P-acquiring extracellular enzyme activities across two contrasting systems: Native Rangeland (mixed prairie vegetation) and Introduced Pasture [ Cynodon dactylon (L.) Pers.]. Two grazing strategies were compared: Adaptive (AD; higher stocking density with variable rest periods) and Prescriptive (PR; lower stocking density with fixed rest periods) across three years. Strong temporal variation was evident across genes and enzyme activities, with greater values in 2022 compared to 2023 and 2024. Overall, grazing affected the nitrate reduction and denitrification pathways. In Native Rangeland, the abundance of napA was up to 57% greater under AD, narG up to twofold greater under PR, and nirS showing inconsistent grazing treatment effects. In Introduced Pastures, nirS was up to 57% greater under PR, whereas nirK showed inconsistent responses. Grazing greatly affected enzyme activity in Native Rangeland under AD, with C-enzymes up to 175%, N-enzymes up to 271%, and P-enzymes up to 216% compared to PR. In Introduced Pastures the greatest differentiation was for N-enzymes, with PR exhibiting two-fold the activity of AD. Overall, our results show that grazing mediates microbial nutrient acquisition in a context-dependent manner. These findings reinforce the importance of accounting for site-specific conditions when applying grazing strategies and interpreting soil microbial responses.