Secondary Metabolites from Australian Lichens Ramalina celastri and Stereocaulon ramulosum Affect Growth and Metabolism of Photobiont Asterochloris erici through Allelopathy

In the present work, the phytotoxic effects of secondary metabolites extracted from lichen Ramalina celastri (usnic acid) and lichen Stereocaulon ramulosum (a naturally occurring mixture of atranorin and perlatolic acid, approx. 3:1) on cultures of the aposymbiotically grown lichen photobiont Asterochloris erici were evaluated. Algae were cultivated on the surface of glass microfiber disks with applied crystals of lichen extracts for 14 days. The toxicity of each extract was tested at the two selected doses in quantities of 0.01 mg/disk and 0.1 mg/disk. Cytotoxicity of lichen extracts was assessed using selected physiological parameters, such as growth (biomass production) of photobiont cultures, content of soluble proteins, chlorophyll a fluorescence, chlorophyll a integrity, contents of chlorophylls and total carotenoids, hydrogen peroxide, superoxide anion, TBARS, ascorbic acid (AsA), reduced (GSH) and oxidized (GSSG) glutathione, and composition of selected organic acids of the Krebs cycle. The application of both tested metabolic extracts decreased the growth of photobiont cells in a dose-dependent manner; however, a mixture of atranorin and perlatolic acid was more effective when compared to usnic acid at the same dose tested. A higher degree of cytotoxicity of extracts from lichen S. ramulosum when compared to identical doses of extracts from lichen R. celastri was also confirmed by a more pronounced decrease in chlorophyll a fluorescence and chlorophyll a integrity, decreased content of chlorophylls and total carotenoids, increased production of hydrogen peroxide and superoxide anion, peroxidation of membrane lipids (assessed as TBARS), and a strong decrease in non-enzymatic antioxidants such as AsA, GSH, and GSSG. The cytotoxicity of lichen compounds was confirmed by a strong alteration in the composition of selected organic acids included in the Krebs cycle. The increased ratio between pyruvic acid and citric acid was a very sensitive parameter of phytotoxicity of lichen secondary metabolites to the algal partner of symbiosis. Secondary metabolites of lichens are potent allelochemicals and play significant roles in maintaining the balance between mycobionts and photobionts, forming lichen thallus.