PCR for T-RFLP profiling was conducted after denaturing for 2 min at 98
C,
followed by 30 cycles of 30 s at 95
C, 45 s at 54
C, and 90 s at 72
C. Aliquots of
the amplified fragments were then separately digested with the restriction enzymes
AluI, HhaI, and HaeIII (Takara Bio) according to the manufacturer’s instructions.
Lengths of T-RFs were determined with a 3130xl DNA Sequencer (Applied
Biosystems, Foster City, CA, USA) by mixing 2 μL of purified T-RF DNA with
15 μL of Hi-Di formamide and 0.1 μL of DNA standard LIZ600 (Applied
Biosystems). Samples were denatured at 96
C for 2 min and immediately chilled
on ice prior to electrophoresis using an ABI automated sequence analyzer. Lengths
of fluorescently labeled T-RFs were determined after electrophoresis by comparison
with internal standards using GeneMapper software (version 3.7, Applied
Biosystems). T-RFLP peaks ranged in size from 50 to 650 base pairs.
Samples with the dominant T-RF peaks were selected for identification by clone
library analysis. T-RF solutions were ligated using the pGEM-T Easy Vector
(Promega, Madison, WI, USA), and Escherichia coli DH5α high efficiency competent cells were used as hosts for recombinant plasmids and grown at 37
C in LB agar
(Merck KGaA, Darmstadt, Germany), to which 100 μg mL
À1 of ampicillin, IPTG,
and Xgal had previously been added to a final concentration of 40 μg mL
À1 . White
colonies were selected, and sequences were determined using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and read on an Applied
Biosystems 3130xl Genetic Analyzer. The primer M13 Primer RV was used in
sequencing reactions to obtain partial DNA sequences. DNA sequences were aligned
using MEGA version 5 software (Tamura et al. 2011), and all sequences were
compared to those in the NCBI database using the BLAST program.
2.2.2 Results and Discussion
2.2.2.1 Sclerotia Grain Density
Sclerotia from Mt. Chokai were spherical, black, with a diameter 0.4 to 2.6 mm
(average 1.33 Æ 0.06 mm; n ¼ 54), whereas sclerotia from Mt. Iwaki were black,
some were spherical and some were irregularly shaped, had a relatively large
diameter, ranging from 0.8 to 4.4 mm (average 2.73 Æ 0.11 mm; n ¼ 54). Based
on morphological characteristics, sclerotia from both areas matched the description
given by Trappe (1962): diameter of 0.05–4 mm or greater, jet black color, hard,
smooth, and mostly spherical. The average diameter of Mt. Iwaki sclerotia was
significantly larger than that of Mt. Chokai sclerotia (p < 0.01; Student’s t-test).
According to Matsumoto and Tajimi (1988), sclerotia size reflects different strategies in response to environmental changes. A larger sclerotial diameter may also
indicate a high extractable aluminum content in the soil or exposure to an event that
enriched extractable aluminum (Watanabe et al. 2002, 2004). The number of
sclerotia ranged between 15 and 27 grains (average 19.83 Æ 0.81) per 800 cm
3 at
Mt. Chokai, whereas at Mt. Iwaki sclerotia were more abundant, ranging from 12 to
2 Fungal Communities of Sclerotia Grains from Forest Soils
21
C,
followed by 30 cycles of 30 s at 95
C, 45 s at 54
C, and 90 s at 72
C. Aliquots of
the amplified fragments were then separately digested with the restriction enzymes
AluI, HhaI, and HaeIII (Takara Bio) according to the manufacturer’s instructions.
Lengths of T-RFs were determined with a 3130xl DNA Sequencer (Applied
Biosystems, Foster City, CA, USA) by mixing 2 μL of purified T-RF DNA with
15 μL of Hi-Di formamide and 0.1 μL of DNA standard LIZ600 (Applied
Biosystems). Samples were denatured at 96
C for 2 min and immediately chilled
on ice prior to electrophoresis using an ABI automated sequence analyzer. Lengths
of fluorescently labeled T-RFs were determined after electrophoresis by comparison
with internal standards using GeneMapper software (version 3.7, Applied
Biosystems). T-RFLP peaks ranged in size from 50 to 650 base pairs.
Samples with the dominant T-RF peaks were selected for identification by clone
library analysis. T-RF solutions were ligated using the pGEM-T Easy Vector
(Promega, Madison, WI, USA), and Escherichia coli DH5α high efficiency competent cells were used as hosts for recombinant plasmids and grown at 37
C in LB agar
(Merck KGaA, Darmstadt, Germany), to which 100 μg mL
À1 of ampicillin, IPTG,
and Xgal had previously been added to a final concentration of 40 μg mL
À1 . White
colonies were selected, and sequences were determined using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and read on an Applied
Biosystems 3130xl Genetic Analyzer. The primer M13 Primer RV was used in
sequencing reactions to obtain partial DNA sequences. DNA sequences were aligned
using MEGA version 5 software (Tamura et al. 2011), and all sequences were
compared to those in the NCBI database using the BLAST program.
2.2.2 Results and Discussion
2.2.2.1 Sclerotia Grain Density
Sclerotia from Mt. Chokai were spherical, black, with a diameter 0.4 to 2.6 mm
(average 1.33 Æ 0.06 mm; n ¼ 54), whereas sclerotia from Mt. Iwaki were black,
some were spherical and some were irregularly shaped, had a relatively large
diameter, ranging from 0.8 to 4.4 mm (average 2.73 Æ 0.11 mm; n ¼ 54). Based
on morphological characteristics, sclerotia from both areas matched the description
given by Trappe (1962): diameter of 0.05–4 mm or greater, jet black color, hard,
smooth, and mostly spherical. The average diameter of Mt. Iwaki sclerotia was
significantly larger than that of Mt. Chokai sclerotia (p < 0.01; Student’s t-test).
According to Matsumoto and Tajimi (1988), sclerotia size reflects different strategies in response to environmental changes. A larger sclerotial diameter may also
indicate a high extractable aluminum content in the soil or exposure to an event that
enriched extractable aluminum (Watanabe et al. 2002, 2004). The number of
sclerotia ranged between 15 and 27 grains (average 19.83 Æ 0.81) per 800 cm
3 at
Mt. Chokai, whereas at Mt. Iwaki sclerotia were more abundant, ranging from 12 to
2 Fungal Communities of Sclerotia Grains from Forest Soils
21