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 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 variability across the growing season, we sampled soils in
each plot in May, July, and September of 2016. In each plot we
extracted three 10 x 10 cm organic horizon layers and homogenized them
into a single sample defining this as the OH soil fraction. Next, we
sampled four 5 cm diameter mineral soil cores to a depth of 15 cm
beneath the OH 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
rhizosphere soil from mineral soil samples via the soil-adhesion
method wherein the rhizosphere soil fraction was operationally defined
as soil that remained clung to roots after modest shaking (Phillips
& Fahey, 2005). After removal of roots, all soils were passed
through a 2 mm sieve and stored at -80°C
until further analysis.
To determine the extent to which N fertilization impacts microbial
allocation to extracellular enzymes, we assayed the potential activity
of hydrolytic enzymes that release N (N-acetylglucosaminidase; NAG),
phosphorus (acid phosphatase; AP), and simple carbon (ß-glucosidase;
BG). In addition, we measured microbial allocation to complex C
degrading oxidative enzymes phenol oxidase and peroxidase. Briefly, 1g
of thawed soil was homogenized in 50mM sodium acetate buffer (pH 5.0).
Next, hydrolytic activities were determined using a fluorometric
microplate assay (Gemini XPS, Molecular Devices, USA). with
methylumbelliferone-linked substrates and oxidative enzymes using a
colorimetric microplate assay (Tecan Infinite 200 Pro, Switzerland)
with L-3,4-dihydroxyphenylalanine linked substrates according to
Saiya-Cork et al., 2002.
Saiya-Cork KR, Sinsabaugh RL, Zak DR (2002) The effects of long term
nitrogen deposition on extracellular enzyme activity in an Acer
saccharum forest soil. Soil Biology and
Biochemistry, 34, 1309–1315. DOI:
10.1016/S0038-0717(02)00074-3.
See protocol:
https://allison.bio.uci.edu/protocols/fluorimetricenzymeprotocol.pdf
