Inhibition of mycelial growth of F. fujikuroi by isolated yeasts
was tested according to Zhang et al. (2010b) with minor modifications.
For the dual culture assay (F. fujikuroi + yeast), a loop of
2-day-old yeast cell suspension grown on NYDA (25 C) was
streaked on a 90 mm diameter Petri Dish with PDA at 30 mm from
the border. A 8 mmmycelium disk of F. fujikuroi, grown on Komada
(1975) at 25 C for 5 days was put at 26 mm from the opposite border
and at 26 mm from the yeast streak. For single culture assay, a
mycelium disk of F. fujikuroi was put on PDA plate as a control.
Three replicates were used both for dual and single culture assays.
Plates were incubated at 25 C for 10 days for daily observations of
the mycelial growth of F. fujikuroi. Inhibition of F. fujikuroi growth
was measured as percentage inhibition of radial growth (PIRG) following
the formula (Fokkema, 1976): R1 R2/R1 100, where R1
corresponds to the radial growth of the fungus towards opposite
site, and R2 corresponds to the radial growth of the fungus towards
the antagonist.
The biocontrol efficacy of the yeast strains was also tested on
seeds naturally infected by F. fujikuroi in Petri dishes. Rice seeds
were immersed in 1% NaOCl for 5 min, washed under tap water,
and air-dried. Cell suspensions for seed treatment were prepared
by growing the selected yeast strains in YEMS (Spadaro et al.,
2010) at 25 C for 48 h on a rotary shaker (200 rpm). Cells were collected
by centrifugation at 5000g for 10 min, re-suspended in Ringer
solution, quantified with a Bürker chamber to reach a standard
concentration of 108 cells ml1. The seeds were then immersed in
the antagonist suspension on a rotary shaker at 90 rpm for
15 min, air dried, and placed in Petri dishes with Komada medium.
Twenty-five seeds were placed per Petri dish and three dishes were
prepared per treatment. The dishes were kept at 25 C for 5 and
10 days to evaluate visually and under light microscope the infection
rate by F. fujikuroi. The biocontrol efficacy test on seeds was
carried out twice.