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.
Evaluation of Trichoderma isolates on wet filter paper:
Based on in vitro antagonism test, 20 Trichoderma isolates
were selected. Rice seeds were soaked in spore suspension of
B. oryzae (10
5
spores mL
-1
) containing 0.05% Tween 20 for 24
h. Then, seeds were air-dried and soaked in spore suspension of
Trichoderma isolates (10
8
spores mL
-1
) containing 0.05%
Tween 20 for 2 h and then placed on the sterile wet filter paper
in a seedling tray with 100 seeds per treatment (7). Plant
height, root length and the percentage of disease control were
determined. The number of infected seedlings was counted for
each treatment and then compared to infected control with B.
oryzae.
Glasshouse experiments
Seed treatment: According to wet filter paper test, 11
Trichoderma isolates were selected and used to inoculate
infested rice seeds with pathogen and were sown in plastic pots
containing autoclaved rice field soil. The pots were kept in
glasshouse with the condition of 21 to 30
°C temperature and
90% relative humidity. Three pots were used for each
treatment. Rice seedlings growth parameters namely stem
height, root length, stem and root wet weight, stem and root dry
weight and percentage of disease control were determined 45
days after inoculation. Rice stem and root were dried in an
oven at 80
°C for 24 hours to a dry weight.
Foliar spray
Twenty one days old rice seedlings (cv. Tarom) were
sprayed with spore suspension of 11 selected Trichoderma
isolates (10
8
spore mL
-1
) with 0.05% Tween 20 in greenhouse.
After 24 h, the plants were sprayed with the spore suspension
of B. oryzae (10
5
spore mL
-1
). The seedlings which were
sprayed with B. oryzae alone, served as control. Observations
on the disease severity were recorded seven days after