the profile (Ah1/Ah2/E/Bs). The IWK profile is a Fulvic Andosol located on
Mt. Iwaki, Aomori Prefecture, at an elevation of 800 m (40
39
0 N, 140
16
0 E;
F. crenata forest, Ah1/Ah2/BA/Bw). Sclerotia grains were collected from each
horizon of the soil profiles.
Sclerotia grains larger than 1 mm in diameter were collected from air-dried soils
using sterilized tweezers. Grains smaller than 1 mm in diameter were collected by
using the floatation method in distilled water. Sclerotia grain samples were exposed
to ultrasonic treatment for 5 min to remove soil from their surface and then dried at
room temperature (20–25
C). The sclerotia grains were weighed using an electronic
balance and their morphological features were observed using a digital high-density
video microscope (VH-7000, Keyence, Osaka) and a scanning electron microscope
(JSM 6490 LA, JEOL, Tokyo). Samples were fractionated into three grain sizes:
large (>2 mm), medium (1–2 mm), and small (<1 mm). Soil samples for
14 C age
determination were prepared by removing sclerotia grains larger than 0.2 mm in
diameter.
The sclerotia, humic acid, and soil humin fractions used for determining the
AMS14
C date were prepared as follows.
Sclerotia: Distilled water was added to untreated air-dried soil and grains were
collected by the flotation method. The grains were then subjected to an ultrasonic
bath for 5 min, followed by drying at 105
C for 24 h. Sclerotia were divided into
three categories, large (>2 mm), medium (1–2 mm), and small (<1 mm),
according to their diameter size. Acid and acid/alkaline treatments were not
conducted since they can damage sclerotia structure.
Humic acid: Any visible sclerotia and plant residues were removed under direct
vision, and the resulting air-dried soil was passed through a 0.2 mm sieve. Humic
acid was extracted according to the methods of Nakamura and Nakai (1988) and
Okuno (1995). The extract was then freeze-dried to obtain a powder sample.
Humin: Any visible sclerotia and plant residue were removed under direct vision,
and the resulting air-dried soil was passed through a 0.1-mm sieve and humin was
repeatedly heat extracted using a solution containing a mixture of 0.1 M NaOH
and 0.1 M Na 4 P 2 O 7 at a 1:1 ratio. The extract was then washed with HCl, washed
with distilled water, and then freeze-dried to obtain a powder sample (Nakamura
and Iwahana 1990).
Total carbon (TC) and humic acid contents of sclerotia were measured by using
the dry combustion method using a SUMIGRAPH NC-22A (Sumika Chemical
Analysis Service Center, Ltd., Osaka, Japan). Five medium-sized air-dried sclerotia
from the ONT soil samples were measured; five replicates were used, and each soil
horizon was measured three times. Measurements of humic acid were taken three
times and the soil was measured four times. Medium-sized sclerotia collected from
Mt. Iwaki soil samples (Iw-1, Iw-2) were also measured, with three replicates.
Accelerator mass spectrometry (AMS) measurements of
14 C/
12 C ratios were
carried out at the Micro Analysis Laboratory, using the 5 MV Tandem accelerator,
at the University of Tokyo, according to Matsuzaki et al. (2004). As 1 mg of carbon
is required for AMS measurements, the number of grains applied for each AMS
7 Dating of Sclerotia Grains in Andosol Profiles
121
Mt. Iwaki, Aomori Prefecture, at an elevation of 800 m (40
39
0 N, 140
16
0 E;
F. crenata forest, Ah1/Ah2/BA/Bw). Sclerotia grains were collected from each
horizon of the soil profiles.
Sclerotia grains larger than 1 mm in diameter were collected from air-dried soils
using sterilized tweezers. Grains smaller than 1 mm in diameter were collected by
using the floatation method in distilled water. Sclerotia grain samples were exposed
to ultrasonic treatment for 5 min to remove soil from their surface and then dried at
room temperature (20–25
C). The sclerotia grains were weighed using an electronic
balance and their morphological features were observed using a digital high-density
video microscope (VH-7000, Keyence, Osaka) and a scanning electron microscope
(JSM 6490 LA, JEOL, Tokyo). Samples were fractionated into three grain sizes:
large (>2 mm), medium (1–2 mm), and small (<1 mm). Soil samples for
14 C age
determination were prepared by removing sclerotia grains larger than 0.2 mm in
diameter.
The sclerotia, humic acid, and soil humin fractions used for determining the
AMS14
C date were prepared as follows.
Sclerotia: Distilled water was added to untreated air-dried soil and grains were
collected by the flotation method. The grains were then subjected to an ultrasonic
bath for 5 min, followed by drying at 105
C for 24 h. Sclerotia were divided into
three categories, large (>2 mm), medium (1–2 mm), and small (<1 mm),
according to their diameter size. Acid and acid/alkaline treatments were not
conducted since they can damage sclerotia structure.
Humic acid: Any visible sclerotia and plant residues were removed under direct
vision, and the resulting air-dried soil was passed through a 0.2 mm sieve. Humic
acid was extracted according to the methods of Nakamura and Nakai (1988) and
Okuno (1995). The extract was then freeze-dried to obtain a powder sample.
Humin: Any visible sclerotia and plant residue were removed under direct vision,
and the resulting air-dried soil was passed through a 0.1-mm sieve and humin was
repeatedly heat extracted using a solution containing a mixture of 0.1 M NaOH
and 0.1 M Na 4 P 2 O 7 at a 1:1 ratio. The extract was then washed with HCl, washed
with distilled water, and then freeze-dried to obtain a powder sample (Nakamura
and Iwahana 1990).
Total carbon (TC) and humic acid contents of sclerotia were measured by using
the dry combustion method using a SUMIGRAPH NC-22A (Sumika Chemical
Analysis Service Center, Ltd., Osaka, Japan). Five medium-sized air-dried sclerotia
from the ONT soil samples were measured; five replicates were used, and each soil
horizon was measured three times. Measurements of humic acid were taken three
times and the soil was measured four times. Medium-sized sclerotia collected from
Mt. Iwaki soil samples (Iw-1, Iw-2) were also measured, with three replicates.
Accelerator mass spectrometry (AMS) measurements of
14 C/
12 C ratios were
carried out at the Micro Analysis Laboratory, using the 5 MV Tandem accelerator,
at the University of Tokyo, according to Matsuzaki et al. (2004). As 1 mg of carbon
is required for AMS measurements, the number of grains applied for each AMS
7 Dating of Sclerotia Grains in Andosol Profiles
121