Improved production of phleichrome from the phytopathogenic fungus Cladosporium phlei using synthetic inducers and photodynamic ROS production by phleichrome

Two different diketopiperazines, cyclo-(l-Pro-l-Leu) and cyclo-(l-Pro-l-Phe), which were isolated from the culture filtrate of Epichloe typhina and found to be inducers of phleichrome production, were chemically synthesized and evaluated for use in the improved production of phleichrome from wild-type and UV-mutagenized strains (M0035) of Cladosporium phlei. When supplemented with PDA and V8 juice agar media, both inducers showed significant increases in the production of phleichrome. Phleichrome production was increased in a dose-dependent manner up to a concentration of maximum yield for both inducers. No further significant induction was observed by supplementing inducers over the concentration of maximum yield. Among the two inducers, cyclo-(l-Pro-l-Phe) showed better inducing capability than cyclo-(l-Pro-l-Leu). The maximum yield was observed from the M0035 strain grown on V8 juice media supplemented with 150 μM cyclo-(l-Pro-l-Phe), which was estimated to be 232.6 mg of phleichrome per gram of mycelia and 10.2 mg of secreted phleichrome per 20 agar-plugs. Interestingly, growth inhibition was observed on V8 juice agar media with 100, 150, and 200 μM cyclo-(l-Pro-l-Phe) but not on PDA with the same amount of inducer, which suggests that the inhibitory effect might be through the overproduction of phleichrome rather than the toxic effect of the inducer itself. Superoxide production by purified phleichrome was dramatically stimulated upon illumination, thus demonstrating photodynamic production of superoxide in vitro by phleichrome.