In order to detect possible differences in N fertilization response between mycorrhizal types, we established a plot network of 6 AM and 6 ECM dominated (>65% diameter at breast height; Table S1) 10 x 10 m plots in the lower elevation hardwood zone of both the reference and fertilized watershed at Bear Brook Watershed, Maine in May 2016 (N=24 plots). Tree species were similar between watersheds with AM trees represented by red maple (Acer rubrum) and sugar maple (Acer saccharum) and ECM trees represented by American beech (Fagus grandifolia), grey birch (Betula populifolia), and yellow birch (Betula alleghaniensis).
To capture fine root biomass and morphology variability across the growing season, we sampled soils in each plot in May, July, and September of 2016 and in June, July, and September of 2017. We sampled four 5 cm diameter bulk mineral soil cores to a depth of 15 cm beneath the organic horizon layer and homogenized these by plot. All samples were kept on ice and transported to West Virginia University for further processing within 48-72 h. Upon return to the lab, we separated and dried all fine roots from bulk mineral soils.
To examine the extent to which N fertilization and recovery drove plant investment in fungal symbiosis belowground, mineral soil roots from the peak growing season sampling date (July) in 2016 and 2017 were examined for mycorrhizal colonization. To determine ECM colonization, fine roots from ECM plots were spread out on a petri dish and root tips were examined under a stereoscopic microscope. Percent ECM colonization was calculated as the percent of total root tips that exhibited visible ECM structures including hyphal mantle and emanating mycelium (Bzdyk et al. 2019).
AM structures are not as clearly visible because they are smaller and can occur within the root. As such, AM roots were first cleared of pigment and stained to determine the presence of AM arbuscules and hyphae. Briefly, roots were cleared of pigment by soaking in 10% potassium hydroxide for 48-96 hours and excess pigment was leached from roots by incubating them in 85% ethanol for 24-48 hours. To remove any remaining pigment, roots were covered in an ammonia/hydrogen peroxide solution for 15 minutes. Finally, roots were acidified for staining in 5% hydrochloric acid and stained for 5-20 minutes in 0.06% trypan blue (Comas et al. 2014). Percent colonization of AM fungi was determined using the grid-intersect method wherein roots were spread across a 1 x 1 cm gridded petri dish and examined with a stereoscopic microscope at each intersect for the presence of arbuscules or hyphae (Giovannetti and Mosse 1980; McGonigle et al. 1990). Percent colonization was calculated as the percent of intersects that were colonized by AM structures.
