Re-investment in plant-microbial interactions after long-term nitrogen fertilization differs by mycorrhizal type in temperate forests

Amplicon sequencing of V4 region of the 16S rRNA gene from soil microbial community DNA. The sampling site is part of the Fernow Experimental Forest located in the Allegheny section of the Appalachian Mountain Range in Tucker County, West Virginia (39.03 N, 79.67 W). We sampled soils from two adjacent watersheds: watersheds 3 and 7. Watershed 3 received elevated nitrogen (N) annually at a rate of 35 kg N ha-1 in the form of ammonium sulfate ((NH4)2SO4) from 1989 to 2019. Watershed 7 (reference) received only ambient N deposition. The tree species found in these watershed predominantly associate with arbuscular mycorrhizal (AM) fungi (e.g. Acer sp. and Prunus serotina), but ectomycorrhizal (ECM) fungi associated trees, such as sweet birch (Betula lenta), red oak (Quercus rubra), and American beech (Fagus grandifolia) are also found throughout the watersheds. In each watershed, we sampled a previously established network of 10 by 10 m AM and ECM dominated plots with six AM and six ECM dominated plots located in each watershed in June 2017 and June 2022 (N = 48 plots total). In each plot, we collected three samples from the top 20 cm of mineral soil using a 5 cm diameter core. The soil samples were homogenized by plot to provide one composite mineral soil sample for each plot. After sieving the samples through a 2 mm mesh, we separated the bulk soil from the soil aggregates adhering to the roots, which we operationally defined as the "rhizosphere" (N = 96 samples total +/- NAs).