Spore–spore confrontation. A conidia suspension of BCA was
prepared by flooding the plates from seven days old culture with
sterile distilled water followed by gently scraping the surface with
a sterile spatula. Concentrations of spore suspensions were individually adjusted in 1.5 mL tubes containing 100 ll of PD broth
medium to 2 105, 4 105, 8 105 and 16 105 cells/mL by use
of a haemocytometer under microscope (HumaScope).
Thereafter, 100 lL of F. solani conidia suspension calibrated at
8 105 cells/mL were introduced in each tube, followed by incubation for 10 and 24 h. Experiments were performed in triplicate.
Tubes containing 100 ll of PD broth medium and 100 lL of F. solani
conidia were considered as negative controls. Aliquots from each
tube were taken and examined microscopically after incubation
to assess spore germination. Percent inhibition of germination
10 R.M.K. Toghueo et al. / Biological Control 96 (2016) 8–20
was determined over the total number of conidia relative to the
negative controls.
2.3.2. Effect of metabolites produced by Trichoderma spp. on the
growth of F. solani
2.3.2.1. Effect of volatile metabolites. The effect of volatile metabolites produced by the BCAs on F. solani mycelial growth was determined as described by Goyal et al. (1994) with little modifications.
The antagonistic fungi were centrally inoculated by placing a 5 mm
diameter mycelial disc taken from 7 days old culture on the PDA
plate and incubated at 25 C for 2 days. The Petri dishes containing
the BCAs were covered with inverted F. solani plates and the plates
sealed together with paraffin tape and further incubated at 25 C
for 7 days. In control treatment, F. solani plates were used to cover
PDA plates without Trichoderma sp. The diameter of pathogen colonies was measured daily during 7 days of incubation. The percent
growth inhibition was calculated as compared to growth in control
plates.