2.2 Analysis of Sclerotia-Associated Fungal Communities
by T-RFLP Profiling
2.2.1 Materials and Methods
Sclerotia were collected from Mt. Chokai in Yamagata Prefecture (39
6
0 42.53
00 N,
139
58
0 0.61
00 E, site elevation: 730 m; annual rainfall: 2362 mm) and Mt. Iwaki in
Aomori Prefecture (40
39
0 0.37
00 N, 140
16
0 33.60
00 E, site elevation: 790 m; annual
rainfall: 1750 mm), both in northern Japan, where sclerotia are known to be
abundant in the soil. Vegetation at the Mt. Chokai site (Andic Podzols, WRB/
FAO-Unesco) is dominated by Fagus crenata, with smaller numbers of Viburnum
furcatum and Lindera umbellata; the forest floor is dominated by Sasa kurilensis.
The Mt. Iwaki site (Fulvic Andosols, WRB/FAO-Unesco) mostly contains Quercus
serrata, F. crenata, and Betula ermanii trees; Sasa kurilensis is the dominant plant
on the forest floor. Within a 10 Â 10 m
2 quadrat, nine evenly distributed points were
selected (n ¼ 9). At each point, a 20 Â 20 cm
2 square was marked from which the
litter, F, and H soil layers were removed, and the A horizon was collected using a
cylinder (10 cm  10 cm), total volume of approximately 800 cm
3 per quadrat.
Sclerotia were collected by hand from the A horizon soil and surface-sterilized using
the following procedure described by Ohta et al. (2003). Briefly, every sclerotia
grain was washed with 1 mL of sterile water in a microtube for 1 min with vortex
mixing, the washing solution was removed with a sterile pipette, and this procedure
was repeated 10 times.
Sclerotia samples were collected from nine quadrat at each study site. Approximately 100 mg of surface-disinfested sclerotia from each study site were placed in a
sterile 2-mL centrifuge tube. A metal crusher was inserted into the tube and 200 μL
of lysis buffer (10 μL Tris-HCl, pH 8, 1 M; 2 μL EDTA, 0.5 M; 10 μL proteinase K;
100 μL SDS 10%, and 878 μL H 2 O) was added and homogenized. The crushed
sclerotia solution was incubated at 37
C with shaking for 2 h. The lysed solution
was extracted using phenol/chloroform, followed by addition of binding buffer (60 g
guanidine thiocyanate; 10 mL Tris-HCl, 1 M; and 40 mL distilled water) and silica.
Guanidine thiocyanate was washed out using wash buffer (10 mM Tris-HCl, pH 7.5;
100 mM NaCl: ethanol ¼ 1: 4). PCR was conducted using the universal primers
ITS1F and ITS4 (Gardes and Bruns 1993) with the iProofTM High-Fidelity PCR kit
(Bio-Rad Laboratories, Hercules, CA, USA) under a hot start at 98
C for 30 s, then
35 cycles consisting of 10 s at 98
C, 30 s at 58
C, and 30 s at 72
C in a thermal
cycler (Takara Bio, Otsu, Japan). PCR products were purified in a Seakem GTGTM
agarose gel (Lonza Group Ltd., Basel, Switzerland) and followed by further purification with the silica method. Purified PCR products were then amplified by
quenching PCR using the quenching-fluorescence-labeled primers qLR21 and
ITS1F. Thirty microliters of the qPCR reaction mixture were prepared by adding
0.1 μg template sclerotia DNA, 1.0 μL of 10 pmol μL
À1 primers, Takara Ex TaqTM
dNTPs, and 3 μL of optimized 10Â Ex buffer (Takara Bio) in a thermal cycler.
20
K. Narisawa et al.
by T-RFLP Profiling
2.2.1 Materials and Methods
Sclerotia were collected from Mt. Chokai in Yamagata Prefecture (39
6
0 42.53
00 N,
139
58
0 0.61
00 E, site elevation: 730 m; annual rainfall: 2362 mm) and Mt. Iwaki in
Aomori Prefecture (40
39
0 0.37
00 N, 140
16
0 33.60
00 E, site elevation: 790 m; annual
rainfall: 1750 mm), both in northern Japan, where sclerotia are known to be
abundant in the soil. Vegetation at the Mt. Chokai site (Andic Podzols, WRB/
FAO-Unesco) is dominated by Fagus crenata, with smaller numbers of Viburnum
furcatum and Lindera umbellata; the forest floor is dominated by Sasa kurilensis.
The Mt. Iwaki site (Fulvic Andosols, WRB/FAO-Unesco) mostly contains Quercus
serrata, F. crenata, and Betula ermanii trees; Sasa kurilensis is the dominant plant
on the forest floor. Within a 10 Â 10 m
2 quadrat, nine evenly distributed points were
selected (n ¼ 9). At each point, a 20 Â 20 cm
2 square was marked from which the
litter, F, and H soil layers were removed, and the A horizon was collected using a
cylinder (10 cm  10 cm), total volume of approximately 800 cm
3 per quadrat.
Sclerotia were collected by hand from the A horizon soil and surface-sterilized using
the following procedure described by Ohta et al. (2003). Briefly, every sclerotia
grain was washed with 1 mL of sterile water in a microtube for 1 min with vortex
mixing, the washing solution was removed with a sterile pipette, and this procedure
was repeated 10 times.
Sclerotia samples were collected from nine quadrat at each study site. Approximately 100 mg of surface-disinfested sclerotia from each study site were placed in a
sterile 2-mL centrifuge tube. A metal crusher was inserted into the tube and 200 μL
of lysis buffer (10 μL Tris-HCl, pH 8, 1 M; 2 μL EDTA, 0.5 M; 10 μL proteinase K;
100 μL SDS 10%, and 878 μL H 2 O) was added and homogenized. The crushed
sclerotia solution was incubated at 37
C with shaking for 2 h. The lysed solution
was extracted using phenol/chloroform, followed by addition of binding buffer (60 g
guanidine thiocyanate; 10 mL Tris-HCl, 1 M; and 40 mL distilled water) and silica.
Guanidine thiocyanate was washed out using wash buffer (10 mM Tris-HCl, pH 7.5;
100 mM NaCl: ethanol ¼ 1: 4). PCR was conducted using the universal primers
ITS1F and ITS4 (Gardes and Bruns 1993) with the iProofTM High-Fidelity PCR kit
(Bio-Rad Laboratories, Hercules, CA, USA) under a hot start at 98
C for 30 s, then
35 cycles consisting of 10 s at 98
C, 30 s at 58
C, and 30 s at 72
C in a thermal
cycler (Takara Bio, Otsu, Japan). PCR products were purified in a Seakem GTGTM
agarose gel (Lonza Group Ltd., Basel, Switzerland) and followed by further purification with the silica method. Purified PCR products were then amplified by
quenching PCR using the quenching-fluorescence-labeled primers qLR21 and
ITS1F. Thirty microliters of the qPCR reaction mixture were prepared by adding
0.1 μg template sclerotia DNA, 1.0 μL of 10 pmol μL
À1 primers, Takara Ex TaqTM
dNTPs, and 3 μL of optimized 10Â Ex buffer (Takara Bio) in a thermal cycler.
20
K. Narisawa et al.