ROOT COLLECTION
Shallow and deep cores were collected at five systematic positions
within each plot, in the central and four corner subplots.
Shallow cores were collected September 22 and October 10, 2010 using a
2” diameter core hammered 10 cm into the soil after removing the Oi
(litter layer). Shallow cores were further separated into organic or
mineral soil horizons, with an average organic layer (Oe and Oa
horizons) depth of 3.2 ± 0.5 cm. Both parts were stored frozen until
analysis. In C5, one core lacked an organic horizon (n = 19); in C7,
all 20 cores included an organic sample. Mineral soil was present in
all but two cores in C5 (i.e., the Oea was > 10 cm). One shallow
core was lost after collection from stand C5.
Deep cores were collected in July 2010 using a gas-powered rotary
corer with a 10-cm diameter diamond-tipped cylindrical drill bit
(Levine et al. 2012). Deep cores were taken 30-50 cm from the top of
the mineral soil for analysis of soil carbon and nitrogen (data not
reported here), which provided an opportunity to characterize roots at
depth. Intact root branches were separated from the soil and frozen
for use in this study. Of the 20 deep cores collected in each stand, 7
root samples from C5 and 4 from C7 were lost and not used in this
analysis. The number of deep cores per plot that provided roots for
this analysis ranged from 2 to 5.
The total number of samples analyzed was 44 in C5: 14 mineral shallow,
17 organic shallow, and 13 mineral deep. In C7 the total number of
samples was 50: 17 mineral shallow, 17 organic shallow, and 16 mineral
deep. For each of these soil core portions, both EM and AM roots were
processed, as described below, for a total of 188 root samples.
ROOT PROCESSING
Distinguishing AM and EM roots
Shallow cores were thawed and washed over a sieve to extract root
branches at least 3 cm in length. Roots from the deep cores were
thawed and washed. All roots were preserved in ethanol until
analysis. Roots were sorted based on morphology and wood anatomy into
AM or EM types viewed under a dissecting microscope (Yanai et al.
2008). Rarely, in later processing steps, a root initially typed as AM
was observed to have a Hartig net and was reclassified as EM. The
mycorrhizal status of AM roots was verified after clearing with
potassium hydroxide and staining with Chlorazol black E, as described
below.
Root Length
To measure the length of AM and EM roots, each root sample was placed
on a dissecting microscope dish and intersections with grid lines were
counted (Newman 1966). Root length per unit soil volume was calculated
by dividing the length of roots by the volume of the core.
Clearing and Staining Roots
Putative AM roots were cleared by autoclaving in 10% potassium
hydroxide solution for 20 minutes at 15 ATM and 120 °C, soaked in 3%
hydrogen peroxide for 10 minutes and washed in 1% nitric acid
(Brundett et al. 1996). Roots were stained in 0.03% Chlorazol black E
and autoclaved for 20 minutes at 15 ATM and 120 °C to reveal fungal
hyphae (Cannon 1941; Brundett et al. 1996). Stained roots were viewed
at 400x to assess the presence of AM features.
Arbuscular Mycorrhizal Colonization
Each sample of cleared and stained AM roots was cut into 2-cm long
segments. Root segments were floated in a Petri dish and one segment
was picked from 25 sections of the dish (Brundett et al. 1996), except
for 70 samples that had only 4-23 segments, all of which were
examined. Root segments were placed on glass slides and examined at
400x magnification, and the presence or absence of the following
mycorrhizal fungal structures was noted at up to 200 intersections of
a micrometer in the eyepiece: coenocytic hyphae, vesicles, arbuscules,
and hyphal coils (McGonigle et al. 1990; Brundett et al. 1996). Roots
were classified as colonized by AM fungi based on the presence of AM
structures, although many other fungal structures were commonly seen
including septate hyphae (evidence of dark septate ascomycete
endophytes) and clamp connections (evidence of basidiomycetes).
Colonization was calculated as the number of intersections with AM
fungal structures divided by the total number of intersections viewed.
Ectomycorrhizal Colonization
To quantify colonization of roots by EM fungi, each root sample was
examined under a dissecting microscope. Colonization was calculated as
the number of root tips colonized by EM fungi divided by the total
number of root tips (Brundett et al. 1996). If necessary, a
cross-section of a root tip was taken for compound microscopy, whereby
the presence of EM features such as a mantle could easily be
distinguished.
