geochronology of glacier fluctuations in the region. (Note, however, that the
“spores” were actually sclerotia; Cg does not produce spores; LoBuglio 1999).
The micromorphological characteristics of Cg sclerotia from the Younger Dryas
in the California Channel Islands have been investigated in terms of their carbon
mineralization (Scott et al. 2010). Carbon spherules from the Younger Dryas have
been identified as fungal sclerotia grains, as described by Scott et al. (2010), and their
investigation has shed light on the cause of the Younger Dryas climate reversal of the
last deglacial warming in the Northern Hemisphere (12,900 year BP). In addition,
evidence of the Younger Dryas impact event is provided by the Lonsdaleite and
nano-diamond particles present inside the carbon spherules (Kinzie et al. 2014).
Daulton et al. (2016) verified that the nano-diamonds are copper spherules in a Cg
sclerotia matrix and that Lonsdaleite-like structures are not hexagonal carbon but
graphene.
Micromorphological observations of the interiors of sclerotia grains were
performed by using electron microscopy to gain a better understanding of the
chemical states of carbon and aluminum in sclerotia grains.
8.2 Materials and Methods
Sclerotia grains were collected from volcanic ash soils in Mount Myoko, Niigata
Prefecture (36
54
0 10
00 N, 138
08
0 15
00 E), Mount Nekodake, Nagano Prefecture
(36
32
0 45
00 N, 138
24
0 00
00 E), and Mount Ontake, Gifu Prefecture (35
55
0 10
00 N,
137
27
0 50
00 E), central Japan. The air-dried grains were sliced in half using a sterilized knife. One half of each grain was coated with carbon for observation under a
scanning electron microscope (SEM, S-800, Hitachi, Tokyo) at a magnification of up
to 100,000Â to observe the morphological features of their interiors. The other half
of each grain was used for determination of elemental composition by energydispersive X-ray (EDX) analysis (PV 9900, EDAX, Mahwah, NJ, USA) at an
accelerating voltage of 15 eV. Quantitative results were obtained using the Super
Quant program of EDAX PV 9900 X-ray analytical software.
Under an optical microscope, samples were collected with a sterilized needle
from the interior of the sclerotia. These power-like samples were scattered on carbon
tape, coated with osmium ion, and analyzed with a thermal field emission SEM
(JSM-7100F or 7800F, JEOL) equipped with an EDX (JED-2300, JEOL), at an
accelerating voltage of 5 kV. Data were analyzed using the ZAF method for
standardless quantitative analysis. The elemental composition of the needle was
also determined, to exclude the possibility of metal contamination from the needle.
Transmission electron microscopy (TEM) was performed using an electron
microscope (200 keV) (JEM-2010F, JEOL, Tokyo). Sclerotia grains collected
from Myoko soil were air dried and then ground into powder and ignited at
550
C for 6 h. Both types of samples were dispersed in CCl 4 using ultrasonic
treatment, placed on a carbon-coated copper grid, and dried. Chemical analysis was
carried out by using an EDX analysis system (EDAX Genesis, Mahwah, NJ, USA).
8 Micromorphological Features of Sclerotia Grains
141
“spores” were actually sclerotia; Cg does not produce spores; LoBuglio 1999).
The micromorphological characteristics of Cg sclerotia from the Younger Dryas
in the California Channel Islands have been investigated in terms of their carbon
mineralization (Scott et al. 2010). Carbon spherules from the Younger Dryas have
been identified as fungal sclerotia grains, as described by Scott et al. (2010), and their
investigation has shed light on the cause of the Younger Dryas climate reversal of the
last deglacial warming in the Northern Hemisphere (12,900 year BP). In addition,
evidence of the Younger Dryas impact event is provided by the Lonsdaleite and
nano-diamond particles present inside the carbon spherules (Kinzie et al. 2014).
Daulton et al. (2016) verified that the nano-diamonds are copper spherules in a Cg
sclerotia matrix and that Lonsdaleite-like structures are not hexagonal carbon but
graphene.
Micromorphological observations of the interiors of sclerotia grains were
performed by using electron microscopy to gain a better understanding of the
chemical states of carbon and aluminum in sclerotia grains.
8.2 Materials and Methods
Sclerotia grains were collected from volcanic ash soils in Mount Myoko, Niigata
Prefecture (36
54
0 10
00 N, 138
08
0 15
00 E), Mount Nekodake, Nagano Prefecture
(36
32
0 45
00 N, 138
24
0 00
00 E), and Mount Ontake, Gifu Prefecture (35
55
0 10
00 N,
137
27
0 50
00 E), central Japan. The air-dried grains were sliced in half using a sterilized knife. One half of each grain was coated with carbon for observation under a
scanning electron microscope (SEM, S-800, Hitachi, Tokyo) at a magnification of up
to 100,000Â to observe the morphological features of their interiors. The other half
of each grain was used for determination of elemental composition by energydispersive X-ray (EDX) analysis (PV 9900, EDAX, Mahwah, NJ, USA) at an
accelerating voltage of 15 eV. Quantitative results were obtained using the Super
Quant program of EDAX PV 9900 X-ray analytical software.
Under an optical microscope, samples were collected with a sterilized needle
from the interior of the sclerotia. These power-like samples were scattered on carbon
tape, coated with osmium ion, and analyzed with a thermal field emission SEM
(JSM-7100F or 7800F, JEOL) equipped with an EDX (JED-2300, JEOL), at an
accelerating voltage of 5 kV. Data were analyzed using the ZAF method for
standardless quantitative analysis. The elemental composition of the needle was
also determined, to exclude the possibility of metal contamination from the needle.
Transmission electron microscopy (TEM) was performed using an electron
microscope (200 keV) (JEM-2010F, JEOL, Tokyo). Sclerotia grains collected
from Myoko soil were air dried and then ground into powder and ignited at
550
C for 6 h. Both types of samples were dispersed in CCl 4 using ultrasonic
treatment, placed on a carbon-coated copper grid, and dried. Chemical analysis was
carried out by using an EDX analysis system (EDAX Genesis, Mahwah, NJ, USA).
8 Micromorphological Features of Sclerotia Grains
141