Agriculture Reference
In-Depth Information
mm x 2m, 80/100 mesh, Shimadzu Good Laboratory Component Center, Tokyo, Japan).
Average CO
2
emission rates were calculated between 0 and 8 h.
Microbial Community Structure Analysis of Tomato Roots by PCR-DGGE
Tomato roots were gently collected from the pots after 30 days of cultivation. DNA was
extracted from a portion (0.1 g) of them using a bead beater (Bead Smash-12, Wakenyaku Co.
Ltd., Kyoto, Japan) with 1 ml of extraction buffer (0.1 M Tris-HCl (pH 8.0), 40 mM EDTA
(pH 8.0), 0.2 M NaCl, 20 g L
-1
SDS) and the supernatant was collected and 400 μl of 7.5 M
NH
4
OAc was added to it and then kept on ice for 5 minutes and centrifuged for 3 min at
13,200 x g and to the supernatant 70 % of volume of ethanol is added and kept in -80ºC for 1
h and centrifuged for 13,200 x g for 10 min and washed with 70 % ethanol and finally
centrifuged and suspended in TE buffer. PCR amplification for DGGE was performed in a 50
μL volume containing 1 μl template DNA, Gene Taq universal buffer (Nippon Gene Co.,
Ltd., Tokyo, Japan), 6.25 nM each dNTP, 30 pmol each primer (F968GC, 1401L2: Muyzer et
al. 1993) and 1.25 U of Gene Taq FP (Takara). The temperature program was as follows: a
denaturing step at 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min,
72°C for 1.5 minn, and final extension step of 72°C for 7 min. DGGE was performed using a
Bio-Rad Dcode
TM
mutation analysis system (Bio-Rad Laboratories, Inc., Tokyo, Japan).
Electorophoresis was done using a 6% (w/v) polyacrylamide gel in 1 x TAE buffer (40 mM
Tris, 20 mM acetic acid and 1 mM EDTA, pH 8.0) under 100 volts at 60°C for 14 h. The
polyacrylamid gels were made with parallel denaturing gradients 40-70% (100% denaturant
contains 7 M urea and 40% formamide).
Chemical and Other Biological Analysis of Pumice
Electric conductivity (EC, pumice:water=1:5), pH (pumice:water=1:2.5), total C and N,
exchangeable cations were measured using the conventional methods (Method of Soil
Analysis). Exchangeable cations were extracted with 1 M ammonium acetate (pH 7.0) and
measured using an atomic absorption spectrophotometer (Z-5010, Hitachi Ltd., Tokyo,
Japan).
Microbial numbers were measured by the direct count method using the ethidium
bromide staining (Roser 1980) and microbial biomass by the chloroform fumigation and
extraction method (Vance et al. 1987).
Biological Control of Bacterial Wilt in Pumice Culture Using A Combination
of Biocontrol Agents and Carbon Substrates
Different biocontrol agents isolated in our laboratory were tested for their disease
suppressing property in combination with different kinds of substrates. Pseudomonas
fluorescens MelRC2Rif (Toyota and Ikeda 1997), P. fluorescens FN2, Rhizobium monglense
CF5b, Aquaspirillum arcticum F3a, Pseudomonas citronellolis F3b, Ralstonia pickettii
