Microorganisms
The antagonistic B. subtilis ABS-S14 used in this investigation was isolated from soil collected from citrus groves around the south of Thailand. It was characterized by Gram staining, cell shape with presence of spores and bio-chemical identification, and its anti-fungal activity was screened on potato dextrose agar (PDA) plates (Sangwanich et al., 2013). B. subtilis was streaked on nutrient agar plates and incubated at 37◦C for 18 h before starting the experiments.
P. digitatum was isolated from decayed mandarin fruit and identified. It was maintained on PDA plates with periodictransfers through mandarin fruit to maintain its pathogenicity(Leelasuphakul et al., 2008). Prior to conducting the experiment the fungus was first transferred to infect sterilized mandarin fruit to maintain its aggressiveness as a pathogen and re-isolated. Asuspension of P. digitatum conidia was prepared from a 7 day old culture. The spore concentration was determined with a haemacytometer using a compound light microscope (40×), and adjusted to104 spores mL−1by dilution with sterilized water.