SUPPRESSIVE MECHANISMS OF USED PUMICE TO BACTERIAL WILT OF TOMATO AND THEIR APPLICATION INTO BIOLOGICAL CONTROL IN HYDROPONIC PUMICE CULTURE - Soil Ecology Research Developments - page 161

Ralstonia solanacearum YU1Rif43 (Toyota and Kimura 1996), Fusarium oxysporum f.
sp. lycopersici race 2 880621 and Pythium aphanidermatum OPU431 were used. The
bacterial strain was overnight cultured at 30C in 10 -1 strength nutrient broth medium (Eiken
Chemical Co., Ltd., Tokyo, Japan). F. oxysporum strain was cultured in Czapek medium
(Dhingra and Sinclair 1995) for three days at 30C and microconidia was obtained by passing
the culture through adsorbent cotton. Pythium strain was cultured for 2 days at 30C on 10 -1
strength PDA agar medium (1/10 PDA) (Eiken Chemical Co., Ltd., Tokyo, Japan) and the
plate with fungal mycelia was directly used as an inoculum.
Cultivation of Tomato in Small Pot Experiments
Pumice (100g dry basis) was put into a vinyl pot 9 cm in diameter and inoculated with
bacterial wilt pathogen at a density of 5 x 10 4 cfu g -1 pumice. In case of Fusarium disease,
pumice was added at a density of 1 x 10 5 spores g -1 pumice and a whole 1/10 PDA plate was
mixed with 100 g pumice for Pythium disease. Unused pumice was added with CaCO 3 at a
rate of 15 g kg -1 . Then, six of two-day- old tomato seeds (Lycopersicon esculentum Mill. cv.
Momotaro) were transplanted into a pot. Pots were prepared in triplicates per treatment and
grown in a Biotron (LPH200, Nippon Medical and Chemical Instruments Co., Ltd., Osaka,
Japan; day:night=12h:12h, 285 µmol m -2 s -1 (photon flux), at 30C. Watering was done two
times a day using Otsuka liquid fertilizer A formula [Otsuka Chemical Co., Ltd., Osaka,
Japan; pH 3.8, N 260, P 120, K 405, Ca 230, Mg 60, Mn 1.5 B 1.5, Fe 2.7, Cu 0.03, Zn 0.09,
Mo 0.03 (mg L -1 )] to adjust its moisture content to pF 2.0 (nearly field capacity). The number
of wilted plants was recorded at 2-day intervals until 30 days after seeding on the following
basis: 0; no wilting symptoms, 1; 0 to 25 % of plant showing wilting, 2; 26 to 50%, 3: 51 to
75%, 4: 76 to 100%.
Measurement of R. Solanacearum YU1Rif43 in Pumice
Since the pathogen was resistant to rifampicin, the number of YU1Rif43 was counted by
the dilution plate method using 10 -1 strength nutrient agar supplemented with cycloheximide,
rifampicin and polymyxin B at a concentration of each 100 mg L -1 .
Substrate Induced Respiration Method to Estimate Dominant Microbes in
Pumice
Bacterial and fungal contribution to total microbial biomass was separated using
antibiotics (Anderson and Domsh 1973). Preincubated used pumice for 7 days at 30C at pF
2.0 was dispensed into a 30 ml glass vial in triplicates and then amended with either each 500
µg g -1 soil of rifampicin and kanamycin or 500 µg g -1 soil of cycloheximide for the inhibition
of bacterial and fungal activities, respectively, together with 2 mg g -1 soil of glucose and 0.5
mg g -1 soil of asparagines. CO 2 emission was measured at 0, 2, 4, 6 and 8 h after addition
using a TCD-GC (GC-8A, Shimadzu, Kyoto, Japan), equipped with a Porapaq Q column (3