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Many complex, natural landscapes have been transformed into simpler agroecosystems by continuous cropping and the application of glyphosate and fertilizers. The current mosaic with different land cover types can harbor different soil microbial communities. Here, we investigated how the microbial community and ryegrass plants responded to glyphosate and nitrogen application to soils from four different cover types (soybean monocropping; wheat/soybean-field pea rotation, herbaceous- and woody- uncropped margins). We predict that soils from different land cover types will display different responses of their detritivore microbial communities and plant growth to herbicide and fertilizer application. All the studied variables differed among land cover types. Woody margins tripled the soil carbon content and the microbial respiration of herbaceous margins, although the later had more diverse soil microbial communities. Soils from soybean monoculture had higher respiration rates (37%) than those from crop rotation, where ryegrass accumulated lower biomass (12% lower). Despite these differences between land cover types, neither glyphosate nor nitrogen fertilization significantly influenced these properties. Only ryegrass plants growing on glyphosate-treated soils accumulated more biomass than the rest whereas they did not respond to nitrogen. These results suggest that glyphosate and nitrogen at recommended doses did not have a significant impact on the soil performance of the different types of cover. • Different land covers promote environmental and microbial heterogeneity in arable soils. • Soil microbes and ryegrass growing in monocropping soils outperformed those from crop rotation. • Microbial respiration and composition from different land covers were not affected by glyphosate and nitrogen addition. [ABSTRACT FROM AUTHOR]