Antifungal Metabolites and Siderophore Production Assays

Production of volatile antifungal metabolites by the BCA was tested on FMEA as described by Payne et al. (2000) (link). Briefly, FMEA plates were inoculated with the BCA by evenly streaking cells and/or spores from a 7-day old culture onto the whole surface of the agar. These cultures were grown at 28°C in dark for 14 days. At this time, plates of the same medium were inoculated with an actively growing T. punctulata mycelial plug (5-mm in diameter). The lids were removed and the plates containing the pathogen were inverted over the BCA plates. The two plate bases were taped together with a double layer of Parafilm (American National Can TM, Greenwich, CT, United States). Control plates were prepared in the same way except that a non-inoculated FMEA plate was used instead of a plate containing the BCA. After a further 7 days of incubation, the colony diameter of T. punctulata growing in the presence of the BCA was measured and compared to that of the control.
Hydrogen cyanide (hydrocyanic acid) production by the BCA was detected as described by Bakker and Schippers (1987) (link). The change in color from yellow to orange–brown on the filter paper impregnated with 0.5% picric acid and 2% sodium carbonate indicated the production of cyanide.
Plates of chrome azurol S (CAS) agar developed by Schwyn and Neilands (1987) (link) and modified by Alexander and Zuberer (1991) (link) known as modified M9 agar, were inoculated with the BCA and incubated at 28°C in dark for 10 days. Development of yellow–orange halo zone around the culture was considered as positive for siderophore production (Alexander and Zuberer, 1991 (link)).