The antifungal tests of essential oils were carried out for assessing its contact and volatile phase effects towards mycelial growth of B. cinerea as described previously (Soylu et al., 2006). For determi- nation of contact effects, the essential oils were dispersed as an emulsion in water using ethanol and Tween 20 (0.1% v/v) and added to PDA immediately before it was emptied into the glass Petri dishes (90 × 20 mm in diameter) at a temperature of 40–45 °C. The concentrations tested were 0.4 to 25.6 μg/ml. The controls received the same quantity of ethanol and Tween 20 mixed with PDA. B. cinerea was inoculated immediately by plating in the centre of each plate with a 7 mm diameter disc of the fungus, cut with a sterile cork borer from the edge of actively growing cultures on PDA plates. The Petri dishes were incubated in the dark at 22 °C. For determination of volatile phase effects, glass Petri dishes (90 × 20 mm, which offer 80 ml air spaces after addition of 20 ml agar media) were used. The Petri dishes inoculated as described above at different concentrations of essential oils were added to sterile filter papers (10 mm diameter, Whatman no.1) and placed on the inner surface of the lid of Petri dishes to obtain final concentrations of 0.05 to 1.6 μm/ml air. The Petri dishes were sealed immediately with parafilm to prevent loss of essential oil vapours and incubated at 22 °C. The mean radial mycelial growth of the pathogen was determined by measuring the diameter of the colony in two directions at right angels when the plate surface of the control Petri was covered by fungus 7 days after inoculation. The fungistatic–fungicidal nature of essential oils was tested by observing revival of growth of the inhibited mycelial disc following its transfer to non-treated PDA. A fungicidal effect was where there was no growth, whereas a fungistatic effect was where temporary inhibition of microbial growth occurred. The agar discs of B. cinerea, which failed to grow were either transferred onto agar media without oils (for contact phase effect of oils) or onto lids of the plate containing ethanol and Tween 20 (0.1% v/v) without oil (for volatile phase effect of oils). Petri plates were incubated for 5days. Activity of the each concentration of the various oils was considered fungicidal if the pathogen did not grow or fungistatic if the pathogen growth occurred.
For each concentration, five replicate plates were used. The mean growth values were obtained and then converted in to the inhibition percentage of mycelial growth in relation to the control treatment by using the formula, MGI(%) = ((dc − dt) / dc) × 100, dc and dt represent mycelial growth diameter in control and treated Petri plates, respectively. The experiments were conducted twice.