Based on results of a former study in 2005, this investigation aimed at quantifying UV- and cold temperature stress-induced changes within the secondary metabolite production of the cultured mycobiont of the lichen Heterodea muelleri (Hampe) Nyl. The chemical profiles of the mycobiont cultures and the lichen thallus were determined by high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) analyses. The voucher specimen of H. muelleri produced diffractaic acid as a major polyketide and barbatic acid as a satellite compound, whereas the untreated mycobiont did not contain any detectable secondary metabolites. While UV-C stress caused a general increase in substance formation, cold temperature stress resulted in a strong activation of barbatic acid biosynthesis. A further series of experiments focused on the effect of diffractaic and barbatic acids on the growth of the symbiotic photobiont Trebouxia jamesii; usnic acid was similarly tested. Pure substances were obtained from mycobiont cultures by performing preparative TLC. A determined quantity of algae was incubated on BBM plates that contained three different concentrations of the pure lichen metabolites. The growth of the photobionts was monitored over a period of 1 mo to evaluate the impact of each substance on the cultured algae. While diffractaic and usnic acid had no noticeable effect, barbatic acid strongly inhibited algal growth and resulted in cell death.
The mycobionts isolated from selected species of Haematomma (Haematomma africanum, Haematomma fenzlianum, Haematomma flourescens, Haematomma persoonii, Haematomma stevensiae) have been successfully cultured. The chemical profile of the mycobionts could be effectively influenced and modulated by varying the composition of the nutrient medium using alternative carbohydrates (glucose, sucrose, and polyols). Under artifical laboratory conditions and simulated environmental stress (exposure to UV light, desiccation, and lower temperatures) the mycobionts began producing typical secondary lichen metabolites after an incubation time of 5-6 months. Modified Lilly and Barnett medium (LBM) and Murashige Skoog Medium favoured the production of depsides such as sphaerophorin and isosphaeric acid. Surprisingly, the mycobiont from H. stevensiae in modified MS medium produced two anthraquinones in the mycelia, haematommone at the base and russulone in the upper parts of the mycelium. By contrast, the natural lichen only produced these anthraquinones in the reddish orange apothecia. The mycobiont from H. flourescens only produced the expected lichexanthone in LBM, enriched with the polyols, sorbitol and mannitol. Once the media requirements and environmental stress factors that trigger polyketide production in lichen mycobionts have been determined, it is possible to obtain a particular lichen product by a completely defined procedure. Using such knowledge, we should be able to study polyketide expression in mycobionts under optimized culture conditions for various genetic applications.
A strain of the lichen mycobiont isolated from a thallus of Parmotrema reticulatum was cultured axenically on different media. The morphology, anatomy, growth of the colonies, and metabolite production were studied. The isolated fungal colonies developed well and showed a remarkable morphogenetic capacity on most of the assayed solid media, e.g., malt extract 2%-yeast extract 0.2% (MEYE), malt extract 1%-yeast extract 0.4%-sucrose 10% (MY10), and the original Lilly & Barnett medium (LB). The identity of the isolated fungus was confirmed by its ITS rDNA-sequence. Atranorin, the major cortical lichen depside, was produced when the colonies were grown over 5 and 10 months on solid LB medium, combined with a dessication treatment. Atranorin was identified by matching of UV spectra obtained from HPLC running and a reference substance in a spectrum library. Colonies grown on MEYE and MY10 with a dessication treatment did not produce any lichen secondary metabolite. Mycobionts grown for 5 months on solid MEYE without a dessication treatment produced triacylglycerides as the major metabolites, and the fatty acids were characterized as their methyl esters. Analysis by TLC and HPLC-DAD of extracts of colonies grown on LB and MY10 without dessication revealed that the typical secondary compounds of the natural lichen were not produced. The major metabolites of the natural lichen thallus were identified by chromatographic and spectroscopic methods.
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