mycelial disks of either B. oryzae and Trichoderma isolates 10
mm away from the edge of the plate opposite to each other.
Plates inoculated with B. oryzae alone served as control. Plates
were incubated at 26 ± 1°C for seven days (12 h light/12 h
darkness). The linear growth was measured. Three replicate
plates were done for each treatment. The percentage of growth
inhibition was calculated using the equation RI= 100 x (R2 -
R1) / R2. Where RI was the percentage of reduction in
mycelial growth, R1 was the averaged growth of pathogen in
treated plates and R2 was the averaged growth of pathogen in
control plates (12).
Production of volatile compounds: The bottoms of Petri
dishes containing PDA were separately inoculated with
mycelial plugs of pathogen and Trichoderma isolates. Then
two bottoms held together with pathogen at top, sealed with
adhesive tape and incubated at 26 ± 1°C in the dark for seven
days. The radial growth of the pathogens was measured. Three
replicates of each treatment were used (11).
Production of non-volatile compounds: A piece of
autoclaved cellophane was placed on the surface of PDA
medium in a 9 cm plate then a 7 mm disk of Trichoderma
isolate was inoculated on the cellophane. The plates were
incubated at 26 ± 1
°C for 72 h. After the incubation period, the
cellophane and the adhering Trichoderma mycelia were
aseptically removed and the centre of each dish was inoculated
with a 5 mm diameter disk of B. oryzae taken from an actively
growing colony. The plates were incubated at 26 ± 1
°C for a
further five days. The control treatment was B. oryzae grown
on PDA plate where previously there was a cellophane disc
without antagonist. Three replicate plates were done for each
treatment. The percentage of growth inhibition was calculated
(12).
Mycoparasitism test: The slide culture method (28) was
used to investigate the mycoparasitic nature of Trichoderma
isolates against B. oryzae. A microscope glass slide covered
with a thin layer of PDA in the agar plate was inoculated with
5 mm mycelial disks of Trichoderma spp. and pathogen, 1 cm
apart from each other. All paired cultures incubated at 26 °C
and regions where the hyphae of Trichoderma isolates met the
hyphae of the pathogen were periodically observed under a
light microscope.