Supplementary file 1_Contrasting spring-fall microclimate and summer nutrient controls drive seasonal soil microbial biomass responses to nitrogen addition and grazing in a temperate meadow steppe.docx

IntroductionUnderstanding the season-dependent effects of land-use change and global change drivers on soil microbial biomass is critical for accurately estimating grassland carbon (C) sequestration, sustaining ecosystem services, and enhancing landscape socio-ecological resilience. However, how season modulates the responses of soil microbial biomass to nitrogen (N) addition, grazing (G), and their interaction (NG) remains poorly characterized.
MethodsWe carried out an experiment with N, G and N addition plus grazing (NG) treatments in temperate meadow steppe located in the easternmost of the Eurasian steppe. We measured soil microbial biomass (including total biomass, bacterial biomass, fungal biomass and fungal-to-bacterial biomass ratio), along with a suite of other biotic and abiotic factors across three seasons (spring, summer and fall) over a two-year period.
ResultsOur results revealed distinct seasonal patterns in both environmental factors and microbial responses. Soil temperature and moisture peaked in summer, while soil dissolved inorganic N was highest in spring. Total microbial biomass, bacterial biomass, and fungal biomass exhibited significant seasonal variability in their responses to N and grazing during the second year of sampling. Specifically, compared to the CK plots, the mean values of these indicators were increased in spring and fall but declined in summer under N, G, and NG treatment. Notably, the fungal-to-bacterial biomass ratio showed the strongest response to N addition and grazing in summer, with grazing overriding the effect of N addition in the NG treatment. Grazing significantly reduced the seasonal coefficient of variation (CV) of microbial biomass (p < 0.05), whereas N addition had no significant effect on this CV. The key factors controlling microbial biomass shifted seasonally: soil microclimate (temperature and moisture) dominated in spring and fall (explaining 44%-47% of microbial biomass variation), while soil nutrients (total N, dissolved organic C) and plant biomass became the primary drivers in summer (accounting for 34%-40% of variation).
DiscussionOver all, the responses of soil microbial biomass to N addition and grazing are strongly seasonal dependent, driven by contrasting controls of spring microclimate and summer nutrients. Single-season sampling fails to capture these dynamics, potentially biasing the assessment of disturbance effects and undermining the development of science-based, seasonally adaptive grassland management strategies. Our findings highlight the necessity of multi-season sampling to improve the understanding of microbial biomass responses to environmental changes and provide critical insights for predicting grassland ecosystem functioning under future global change scenarios.