Microbiology, chemistry, ecophysiology of lichen fungi

Recently, highly adapted microbiological methods have been developed that enable large quantities of lichen symbionts to be grown, aposymbiotically and axenically for many possible applications. DNA analyses and the identification of secondary metabolites provide powerful methods for comparing the identity of the isolated mycobionts and the parent lichen specimens. A well documented culture collection of over 150 mycobionts isolated from lichens derived from various climatic regions, chemically interesting specimens (including species of the genera Cladonia, Evernia, Lobaria, Ramalina, Usnea, Umbilicaria, etc.) are used for the novel test series. In culture, many lichen mycobionts exhibit unusual morphogenetic plasticity to form both relatively undifferentiated networks of fungal hyphae and, in addition to mycelia of a considerable morphological complexity. The differentiation processes in lichens and lichen fungi are genetically controlled, but they are also closely correlated with ecological factors. Environmental factors have been found to be critical in the establishment and maintainance of the lichen symbiosis in natural habitats and generally, the survival of lichens in the very diverse ecological environments. From an ecological point of view, lichens have been defined as micro-ecosystems and extremely stress tolerant organisms. Previous studies on lichen mycobiont cultures have shown that external factors, e.g. the type and amount of carbohydrate in the substrates (nutrient media) in combination with stress factors, (e.g. desiccation, temperature changes and different light regimes) affect growth rates, differentiation processes and the production of secondary compounds. In the present work the morphogenesis of the mycelia and the formation of microcrystals (lichen substances) by aerial and medullary hyphae will be studied by ultrastructural methods. Lichen fungi produce interesting biologically active molecules which have considerable potential for use in medicine and pharmacy. Previously such applications have been dependent upon large-scale collections of natural thalli, but these could be reduced or substituted by the use of cultured materials in a near future. In the first part of the proposed project, selected lichen fungi will be cultured under a wide variety of conditions to increase the yields of secondary compounds necessary for applications. Previous investigations have demonstrated that the culturing of mycobionts under constant conditions provided relatively undifferentiated masses of fungal hyphae lacking all the secondary compounds. The second part of the investigation will deal with lichen polysaccharides. Many of such compounds have been screened for pharmaceutical properties and some of them have been found to exhibit significant anti-tumor effects on cancer cell lines. Former studies used extracts from natural lichen thalli. In the planned investigation, heteropolysaccharides, particularly galactomannans formed in mycobiont and photobiont cultures will be identified and anlysed by mass spectrometry. This field has remained largely unexplored since the pioneer studies in the late 1970`s.

Lichens are fascinating, composite plants, forming a symbiosis between ascomycetous fungi and green algae/bluegreen algae (=cyanobacteria). 1-2 % of the lichen fungi are basidiomycetous fungi, forming tiny "mushrooms" living together with green algae and cyanobacteria. The culture collection of the Department of Organismic Biology at the University of Salzburg contains a high number of lichen fungi (mycobionts) and algae/cyanobacteria (photo-, cyanobionts), collected in the different climatic regions of the five continents. Lichen Fungi have been recognised as a rich source of biologically active metabolites since the beginning of the 20th century. However, applications of "lichen substances" have been largely unexplored and unexploited, because mycobionts initially grow very slowly. New nutrient media compositions had to be introduced to obtain higher growth rates and to culture mycobionts and photobionts in bulk quantity. By experimentation and large-scale culture experiments the working group at the University of Salzburg has explored novel nutrient media combinations and culture conditions that allow us to grow mycobionts and photobionts in higher quantities. It was found by "case" studies that most lichen fungi do not form "lichen substances" (secondary metabolites; polyketides) under stable and standardised culture conditions. Electronically adjustable culture chambers - growing mycobionts under varied microclimatic changes - stimulated lichen fungi to start cell differentiation and finally secondary compound production. The polyketide contents were analysed by TLC and HPLC (Thin layer and High performance Liquid Chromatography) For the first time, a considerable number of lichen fungi forming highly differentiated mycelia were found to switch on a particular polyketide pathway by producing the same or related phenolic compounds, typical for the original lichen (from which the fungus was isolated). In the most successful "case" studies, species of the genus Lobaria, Bunodophoron, Ramalina produced complete chemosyndromes (a cohort of chemically related metabolites) in culture. The obtained results were presented as talks and posters on several International Meetings, Conferences, Symposia, Congresses in Europe, USA and Australia. The publication of "Ramalina" was honoured by the "Tuckerman Award 2005" by the ABLS (American Bryological And Lichenological Society, a prize given to the best paper in the American Journal "The Bryologist" (see publications)). Further exciting results were obtained by analyses of the polysaccharides (galactomannanes) from lichens and cultured mycobionts. New lichen heteropolysaccharides were detected and novel chemical structures were elucidated by mass spectroscopy. The collaboration with the research lab of Curitiba (Department of Biochemistry, University of Parana) in Brazil was very successful resulting in several publications in international journals with high impact factors (FEMS, Carbohydrate Research, Phytochemistry, Mycological Research, etc.).