fractions. The sum of recovered bacteria as CFU from sample C was 1.46 Â 10
6
(g fresh weight)
À1 : 88% were found in the combined wash fractions and 12% in the
ultrasonicate. Fungal propagules were counted at 1.47 Â 10
5 (g fresh weight)
À1
(86%) in the wash fractions and 2.40 Â 10
4 (g fresh weight)
À1 (14%) in the
ultrasonicate (Ohta et al. 2003). This experiment shows that, to isolate bacteria
from the inside of sclerotia grains collected from soil, the grains should be washed
at least 10 times.
4.4 Bacterial Colonization of Sclerotia Grains
The experiment described in this section was conducted to determine which types of
bacteria inhabit sclerotia under natural conditions in the field, and whether sterile
sclerotia grains could be colonized with selected bacteria under controlled conditions. Such a process, if successful, would be valuable for introducing beneficial
bacteria to soils by using sclerotia as microbial carrier substrates.
4.4.1 Materials and Methods
4.4.1.1 Soil Sampling and Collection of Ontake Sclerotia
Soil samples were collected from the A horizon of Fulvic Andosol (FAO 2015)
under northern Japanese hemlock trees (Tsuga diversifolia) in the Yunohana Pass
(elevation 2103 m) on Mt. Ontake in Gifu Prefecture, Japan. Sclerotia grains were
collected from the soil samples stored at 4
C by a modification of the wet-sieving
method (Brundrett 1996), using 0.5-, 1.0-, and 2.0-mm sieves.
4.4.1.2 Isolation and Identification of Bacteria Inside Sclerotia
Samples for microbial community analysis were isolated from the insides of four
sclerotia (particle diameters of approximately 2.7 [Sc1], 2.2 [Sc2], 1.6 [Sc3], and 1.2
[Sc4] mm) selected from among all of the sclerotia collected; the wet weights of the
selected sclerotia were 9.2, 6.4, 2.4, and 1.7 mg, respectively. Following the washing
method described in Sect. 4.3, bacteria were isolated from the insides of sclerotia,
cultured on 100-fold diluted nutrient broth (DNB) agar and identified by 16S rRNA
sequencing.
4.4.1.3 Comparison of Sclerotia Before and After Autoclaving
Sclerotia collected from soil samples were sterilized by autoclaving (121
C for
20 min). The internal cellular structure of the sclerotia was observed with an electron
68
Y. S. Nonoyama and K. Narisawa
6
(g fresh weight)
À1 : 88% were found in the combined wash fractions and 12% in the
ultrasonicate. Fungal propagules were counted at 1.47 Â 10
5 (g fresh weight)
À1
(86%) in the wash fractions and 2.40 Â 10
4 (g fresh weight)
À1 (14%) in the
ultrasonicate (Ohta et al. 2003). This experiment shows that, to isolate bacteria
from the inside of sclerotia grains collected from soil, the grains should be washed
at least 10 times.
4.4 Bacterial Colonization of Sclerotia Grains
The experiment described in this section was conducted to determine which types of
bacteria inhabit sclerotia under natural conditions in the field, and whether sterile
sclerotia grains could be colonized with selected bacteria under controlled conditions. Such a process, if successful, would be valuable for introducing beneficial
bacteria to soils by using sclerotia as microbial carrier substrates.
4.4.1 Materials and Methods
4.4.1.1 Soil Sampling and Collection of Ontake Sclerotia
Soil samples were collected from the A horizon of Fulvic Andosol (FAO 2015)
under northern Japanese hemlock trees (Tsuga diversifolia) in the Yunohana Pass
(elevation 2103 m) on Mt. Ontake in Gifu Prefecture, Japan. Sclerotia grains were
collected from the soil samples stored at 4
C by a modification of the wet-sieving
method (Brundrett 1996), using 0.5-, 1.0-, and 2.0-mm sieves.
4.4.1.2 Isolation and Identification of Bacteria Inside Sclerotia
Samples for microbial community analysis were isolated from the insides of four
sclerotia (particle diameters of approximately 2.7 [Sc1], 2.2 [Sc2], 1.6 [Sc3], and 1.2
[Sc4] mm) selected from among all of the sclerotia collected; the wet weights of the
selected sclerotia were 9.2, 6.4, 2.4, and 1.7 mg, respectively. Following the washing
method described in Sect. 4.3, bacteria were isolated from the insides of sclerotia,
cultured on 100-fold diluted nutrient broth (DNB) agar and identified by 16S rRNA
sequencing.
4.4.1.3 Comparison of Sclerotia Before and After Autoclaving
Sclerotia collected from soil samples were sterilized by autoclaving (121
C for
20 min). The internal cellular structure of the sclerotia was observed with an electron
68
Y. S. Nonoyama and K. Narisawa