microscope (Keyence VE-9800), and changes in chemical structure before and after
autoclaving were evaluated by Fourier transform infrared spectrometry (FT-IR,
Shimadzu IR Prestige-21). The presence or absence of bacteria before and after
autoclaving was evaluated by culturing on DNB agar using the washing method
described in Sect. 4.3.
4.4.1.4 Sclerotia Colonization Trial
Pure cultures of eight isolates selected at random from among all of the isolates
cultured from the insides of sclerotia were prepared as pure cultures in liquid DNB
medium and diluted 1000-fold to reduce the influence of the nutrient broth (Mitsui
et al. 1997). Autoclaved sclerotia were immersed in the diluted pure liquid cultures.
The sclerotia were removed at set times (1, 5, 10, 15, 24, 48, and 80 h) and rinsed
10 times with sterile water. The sclerotia were crushed with a glass rod in sterile
water, and bacteria in the resulting suspension were enumerated by using the serial
dilution method on DNB agar to determine maximum colonization and
colonization rate.
4.4.2 Results
4.4.2.1 Comparison of Sclerotia Before and After Autoclaving
Examination of the surface morphological structure of autoclaved sclerotia with an
electron microscope revealed that the structure remained essentially intact and the
external surface appeared to be unchanged, although there was some fracturing of
the external surface and damage to the cellular structure in some sclerotia. The FT-IR
spectral peaks (Fig. 4.4) from before and after autoclaving showed no change. From
these results we conclude that autoclaving does not substantially alter the physical or
chemical structure of sclerotia. No bacteria were detected in suspensions of crushed
autoclaved sclerotia cultured on DNB agar.
4.4.2.2 Isolation and Identification of Bacteria Inside Ontake Sclerotia
Different bacterial communities were isolated from the insides of four sclerotia
recovered from the Ontake soil (Fig. 4.5). Only proteobacteria were isolated from
Sc1, and only actinobacteria with one proteobacteria were isolated from Sc2. In
contrast, species from various groups of bacteria were isolated from Sc3 and Sc4.
The dominant genus and the percentage of isolates in the dominant genus from each
sclerotium were Ralstonia (71%) for Sc1, Arthrobacter (45%) for Sc2, Bacillus
(69%) for Sc3, and Nocardioides (50%) for Sc4.
4 Sclerotia Grains as Bacterial Carriers in Soil
69
autoclaving were evaluated by Fourier transform infrared spectrometry (FT-IR,
Shimadzu IR Prestige-21). The presence or absence of bacteria before and after
autoclaving was evaluated by culturing on DNB agar using the washing method
described in Sect. 4.3.
4.4.1.4 Sclerotia Colonization Trial
Pure cultures of eight isolates selected at random from among all of the isolates
cultured from the insides of sclerotia were prepared as pure cultures in liquid DNB
medium and diluted 1000-fold to reduce the influence of the nutrient broth (Mitsui
et al. 1997). Autoclaved sclerotia were immersed in the diluted pure liquid cultures.
The sclerotia were removed at set times (1, 5, 10, 15, 24, 48, and 80 h) and rinsed
10 times with sterile water. The sclerotia were crushed with a glass rod in sterile
water, and bacteria in the resulting suspension were enumerated by using the serial
dilution method on DNB agar to determine maximum colonization and
colonization rate.
4.4.2 Results
4.4.2.1 Comparison of Sclerotia Before and After Autoclaving
Examination of the surface morphological structure of autoclaved sclerotia with an
electron microscope revealed that the structure remained essentially intact and the
external surface appeared to be unchanged, although there was some fracturing of
the external surface and damage to the cellular structure in some sclerotia. The FT-IR
spectral peaks (Fig. 4.4) from before and after autoclaving showed no change. From
these results we conclude that autoclaving does not substantially alter the physical or
chemical structure of sclerotia. No bacteria were detected in suspensions of crushed
autoclaved sclerotia cultured on DNB agar.
4.4.2.2 Isolation and Identification of Bacteria Inside Ontake Sclerotia
Different bacterial communities were isolated from the insides of four sclerotia
recovered from the Ontake soil (Fig. 4.5). Only proteobacteria were isolated from
Sc1, and only actinobacteria with one proteobacteria were isolated from Sc2. In
contrast, species from various groups of bacteria were isolated from Sc3 and Sc4.
The dominant genus and the percentage of isolates in the dominant genus from each
sclerotium were Ralstonia (71%) for Sc1, Arthrobacter (45%) for Sc2, Bacillus
(69%) for Sc3, and Nocardioides (50%) for Sc4.
4 Sclerotia Grains as Bacterial Carriers in Soil
69