Biosystems). A homology search of DNA sequences was performed with the DDBJblastn program.
2.3.1.3 Isolation of Fungi by Culturing
We conducted culture trials to directly isolate and identify fungi from sclerotia.
Following the method published by Narisawa et al. (1998), after washing the
surfaces of sclerotia with 0.005% Tween 20, we placed individual sclerotia on
artificial growth media to isolate fungi. The three types of growth media used were
cornmeal agar (CMA, 17 g L
À1 ; Difco Laboratories, Detroit, MI, USA), CMA
adjusted with sulfuric acid to pH 5.0, and modified Melin–Norkrans (MMN)
medium with a glucose concentration of 0.5%. Sclerotia were incubated at room
temperature (24
C) for at least 1 month under dark conditions. Hyphae growing
from each sclerotium produced a single-species colony. A total of 38 isolates were
obtained. Hyphae growing out of the sclerotia were transferred to 1/2 CMMY
medium (8.5 g cornmeal agar, 10.0 g malt extract, 2.0 g yeast extract, and
7.5 g L
À1 bacto agar; all from Difco). To identify the isolates, a portion of each
colony was scraped and DNA was extracted with PrepMan Ultra Sample Preparation
Reagent (Applied Biosystems CA, USA). PCR amplification was performed under
the conditions described above. After purification, a sequencing reaction was
performed, and the DNA sequence was analyzed.
2.3.1.4 Isolates’ Ability to Form Sclerotia and Sclerotial Morphology
To investigate isolates’ ability to form sclerotia and sclerotial morphology, isolates
were cultured on three types of media (CMA, MMN, and 1/2 CMMY) at room
temperature (24
C) for approximately 1 month. An isolate of C. geophilum AT353
provided by Dr. A. Yamada obtained from roots was used for morphological
comparison. Slide cultures were made of isolates that formed sclerotia on the
above media, and the morphological characteristics of the sclerotia were observed
under a light microscope (BX51, Olympus, Tokyo, Japan).
2.3.2 Results and Discussion
2.3.2.1 Identification of Isolates by the Clone Library Method
A total of 14 fungal species were identified by the clone library method (Table 2.2).
Of the 45 clones, only one was identified as C. geophilum, which has previously
been reported to form sclerotia in forest soil. Three clones identified as vouchered
mycorrhizal fungi exhibited strong sequence homology to C. geophilum. None of
26
K. Narisawa et al.
2.3.1.3 Isolation of Fungi by Culturing
We conducted culture trials to directly isolate and identify fungi from sclerotia.
Following the method published by Narisawa et al. (1998), after washing the
surfaces of sclerotia with 0.005% Tween 20, we placed individual sclerotia on
artificial growth media to isolate fungi. The three types of growth media used were
cornmeal agar (CMA, 17 g L
À1 ; Difco Laboratories, Detroit, MI, USA), CMA
adjusted with sulfuric acid to pH 5.0, and modified Melin–Norkrans (MMN)
medium with a glucose concentration of 0.5%. Sclerotia were incubated at room
temperature (24
C) for at least 1 month under dark conditions. Hyphae growing
from each sclerotium produced a single-species colony. A total of 38 isolates were
obtained. Hyphae growing out of the sclerotia were transferred to 1/2 CMMY
medium (8.5 g cornmeal agar, 10.0 g malt extract, 2.0 g yeast extract, and
7.5 g L
À1 bacto agar; all from Difco). To identify the isolates, a portion of each
colony was scraped and DNA was extracted with PrepMan Ultra Sample Preparation
Reagent (Applied Biosystems CA, USA). PCR amplification was performed under
the conditions described above. After purification, a sequencing reaction was
performed, and the DNA sequence was analyzed.
2.3.1.4 Isolates’ Ability to Form Sclerotia and Sclerotial Morphology
To investigate isolates’ ability to form sclerotia and sclerotial morphology, isolates
were cultured on three types of media (CMA, MMN, and 1/2 CMMY) at room
temperature (24
C) for approximately 1 month. An isolate of C. geophilum AT353
provided by Dr. A. Yamada obtained from roots was used for morphological
comparison. Slide cultures were made of isolates that formed sclerotia on the
above media, and the morphological characteristics of the sclerotia were observed
under a light microscope (BX51, Olympus, Tokyo, Japan).
2.3.2 Results and Discussion
2.3.2.1 Identification of Isolates by the Clone Library Method
A total of 14 fungal species were identified by the clone library method (Table 2.2).
Of the 45 clones, only one was identified as C. geophilum, which has previously
been reported to form sclerotia in forest soil. Three clones identified as vouchered
mycorrhizal fungi exhibited strong sequence homology to C. geophilum. None of
26
K. Narisawa et al.