120 



Journal of the Kentucky Academy of Science 66(2) 



ington, KY. Fungi were maintained by serial 

 transfer on PDA, typically at 1-4 week inter- 

 vals, although sometimes more frequently. 

 Stock and experimental cultures were main- 

 tained at room temperature, typically 20- 

 22°C. Botnjtis cinerea (BC) and Pestalotia sp. 

 (PESP) stock cultures were commonly main- 

 tained in the dark, although PDA cultures of 

 PESP and CG were sometimes maintained 

 under standard fluorescent lights (12 hr light/ 

 dark and 24 hr light, respectively) to promote 

 spore germination. Spore suspensions of CG 

 and PESP were prepared by agitating small 

 plugs (ca. 3 X 10 mm) cut from sporulating 

 cultures on PDA in sterile distilled water 

 (SDW) with a vortex mixer. Spores of Moni- 

 linia fructicola (MF) were produced by cul- 

 turing the fungus on commercially-available 

 unsulphured dried apricots, which were re- 

 hydrated prior to sterilization by autoclaving. 

 Spores of MF were transferred to a small vol- 

 ume of SDW with a transfer loop, and the 

 suspension was agitated with a vortex mixer. 

 Because the presence or absence of light was 

 otherwise found to have no effect on the out- 

 comes of our experiments, inoculated plates 

 were maintained under either condition, as 

 space permitted. 



Two mixed cultures of bacteria that exhib- 

 ited antifungal activity in preliminary co-cul- 

 ture experiments with BC and PESP were 

 designated as "brown" or "white" based on 

 their appearance in PDA culture. The brown 

 culture was isolated from rose leaves exhibit- 

 ing symptoms of the black spot disease 

 (caused by Diplocarpon rosae, DR) and was 

 found to completely prevent DR spore ger- 

 mination on PDA when present in inoculum 

 drops. This brown culture begins to produce 

 a visually detectable, brown diffusible pigment 

 3-4 days after inoculation to PDA or PDB cul- 

 ture media. The white bacterial culture was 

 obtained as a laboratory contaminant. It also 

 appeared to produce one or more diffusible 

 colorless antifungal substances on PDA. Bac- 

 terial culture fluids (BCF) from prolonged (18 

 days) shake cultivation of the brown and white 

 cultures in PDB (designated BRCF and WCF, 

 respectively) were employed. This lengthy cul- 

 tivation period appeared to have resulted in 

 the death of bacteria, based on the absence of 

 growth from aliquots of culture fluids plated 

 onto PDA. Bacterial cultures were maintained 



by serial transfer on PDA, usually at 1-4 week 

 intervals. 



Assessment of SA Effects on Growth of 

 Fungi on PDA 



Because SA is not stable at autoclave tem- 

 peratures, culture media were amended with 

 SA after autoclaving. Petri plates containing 

 —20 ml of PDA were amended with 200 |jl1 

 of SDW or 200 jjlI of sterile aqueous stock 

 solutions of SA applied to the solidified PDA 

 and allowed to diffuse evenly throughout the 

 agar for 3-4 days. The extent of SA diffusion 

 in these plates was assessed by brief visuali- 

 zation of SA fluorescence under a germicidal 

 UV lamp. Plugs (typically 3-5 mm on a side) 

 were cut from the margin of 3-5 day fungal 

 cultures on PDA and placed in the center of 

 Petri plates. The resultant fungal colonies 

 were measured when the fastest-growing col- 

 onies neared the edges of Petri plates, often 

 after 2-3 days incubation at 20-22°C in the 

 dark. Long and short diameters of the typically 

 ovoid individual colonies were averaged for 

 statistical analyses. 



Detection and Assessment of Synergistic 

 Interactions of SA with Other Antifungal 

 Materials 



Dose-response studies with individual ma- 

 terials were conducted with each of the test 

 fungi employed in these experiments (BC, 

 CG, and PESP) to guide the selection of suit- 

 able doses for interaction studies. For experi- 

 ments conducted on PDA, SA and PQ were 

 added after autoclaving. For experiments con- 

 ducted in 24- well culture plates, aliquots of 

 the liquid GS medium (0.4, 0.5, or 1.0 ml for 

 mycelial growth assays, 0.4 ml for spore ger- 

 mination assays) were added to wells and then 

 amended with stock solutions of antifungal 

 materials. In studies of the interactive effects 

 of SA and PQ on BC growth in wefl-plate cul- 

 ture, incubation continued untfl wefls that 

 supported significant growth of BC were filled 

 with fungal mycelia (typically 15 days). 

 Growth was assessed visually and evaluated as 

 present/strong ( + ), absent ( — ), or weak (±). 

 In studies of SA interactions with Cu or BCF 

 in 24- well plates, fungal growth in well-plate 

 assays was assessed by several techniques, usu- 

 ally after 3-4 days for mycelial growth assays 

 and after 1 day for spore germination assays. 



