S. Sharmin Siddique
Scientific Officer, Plant Pathology Division,RARS, Jessore.
K. A.Bhuiyan
Professor, Plant Pathology Department, Bangabandhu Sheikh Mujibur Rahman Agri-cultural University, Gazipur; Bangladesh
M. R. Uddin
Senior Scientific Officer, Horticulture Division, Regional Agricultural Research Station, Jessore
M. B. Anwar
Scientific Officer, Plant Pathology Division,RARS, Jessore.
Growth factors, In-vitro growth, Fusarium oxysporum f.sp. phaseoli
Variety and Species
Influence of five important growth factors namely temperature during incubation, and pH, nitrogen source, carbon source and C/N ratio of the culture medium were tested following in-vitro method (Dhingra and Sinclair 1985). First two experiments were conducted to test five temperature regimes (15, 20, 25, 30 and 35 C) and ten levels of pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5). In both the experiments, potato dextrose agar (PDA) and potato dextrose broth (Tuite 1969) were used. Another three experiments were conducted to test the effect of five nitrogen sources (NaNO3, NH4NO3, (NH4)2SO4, peptone and L-Asparagine), five carbon sources (Dextrose, D-Xylose, Sucrose, Glycerol and D- mannitol) and six levels of C/N ratio (5, 10, 20, 40, 80, and 100) on mycelium growth of F. oxysporum f.sp. phaseoli. Czapek’s solution with agar (semisolid) and without agar (liquid) was used as basal media. The test fungus was F. oxysporum f.sp. phaseoli which was iso-lated from foot rot infected bush bean seedling following tissue planting method (Tuite 1969). To prepare its inocula, the fungus was grown on PDA medium in Petri dishes. Mycelial discs were cut from growing edge of 4 days old culture of the fungus with a flame sterilized 5 mm diameter cork borer. Except the experiment with pH levels, the pH of the culture medium was adjusted to 6 using 0.1N HCl and 0.1N NaOH and sterilized in an autoclave at 120C under 1kg/cm2 pressure for 20 minutes. The liquid medium (broth) was prepared using all ingredients except agar. To test carbon sources, Czapek’s medium was prepared by mixing each carbon source at 30 g per 1 liter. Czapek’s medium without any carbon source was used as control. To study the influence of different ni-trogen sources, the Czapek’s medium was prepared by mixing requisite quantity of each nitrogen source which is equivalent to the amount of nitrogen obtainable from 3g of NaNO3. The Czapek’s medium without any nitro-gen source was used as a control. To test the effect of C/N ratio on growth of the pathogen, Czapek’s medium was prepared by mixing requisite quantity of NaNO3 as a source of nitrogen at two fixed levels of sucrose (2g and 5g) as a source of carbon. The levels of the C/N ratio were 5, 10, 20, 40, 80, and 100. The growth of the fungus was measured in terms of colony diameter and mycelial dry weight. To measure colony diameter, agar media (semisolid) was used and the fungus was grown in 90 mm glass Petri dishes. For the measurement of mycelium dry weight, the fungus was grown in 100 ml conical flasks contain-ing liquid medium (without agar). The agar media were dispensed into the Petri dishes and the liquid media were also poured into the conical flasks at 20 ml per dish or flask. To measure colony diameter, Petri plates containing agar medium were inoculated after solidification with the mycelium discs (0.5 cm) of the test fungus. The inoculum was placed at the center of each plate. Except the experiment to test different temperature regimes, the inoculated Petri plates under all experiment were incu-bated at 25C in incubators. The plates were arranged in the incubators following completely randomized design with three replications. The colony growth was meas-ured by averaging the two diameters taken at right angle for each colony after 4 days of incubation. To measure mycelium dry weight, conical flaks containing potato dextrose broth or liquid Czapeck’s medium (40 C) were inoculated with the inoculum at one disc per flask. The inoculated flasks were incubated at room temperature (25-28 C) for 14 days. The flasks were arranged on the laboratory desks following completely randomized design with three replications (flasks). At the end of incubation period, the cultures inall flasks were filtered through dry (at 70 C for 12 hr) and pre-weighed filter paper. Dry weight of mycelium was determined after drying the mycelium along with the filter paper in an oven at 70 C for constant weight. Dry weight of mycelium was obtained by subtracting weight of only filter paper from weight of filter paper plus mycelium. Data collected from different experiments were analyzed for ANOVA using MSTAT-C program. Dun-can’s Multiple Range Test (DMRT) was performed to compare treatment means. Whenever necessary, data were transformed following appropriate method before statistical analysis.
Bangladesh J. Plant Pathol. Vol. 28 (1&2): 9-14.
Journal