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Genetic diversity of protoparmeliopsis muralis and selectivity in its photobiont choice

Final Activity Report Summary - MURALIS_PHOTOBIONT (Genetic diversity of protoparmeliopsis muralis and selectivity in its photobiont choice)

Lichens are symbiotic organisms composed of at least two different organisms: fungi (mycobiont) and green algae (photobiont) and/or cyanobacteria. In the project we focused on Protoparmeliopsis muralis that is very common lichen colonising diverse substrata. It was suggested that this lichen exhibits a low selectivity level in its photobiont choice and based on ITS rDNA analysis it was found that the following Trebouxia strains are compatible bionts for this fungus: T. asymmetrica, T. gigantea, T. incrustata and unidentified Trebouxia sp. During the project we successfully isolated and cultured in-vitro mycobiont and photobionts from P. muralis. ITS rDNA analysis showed that the photobionts belonging to unidentified Trebouxia strain (here called 'muralis') form a separate clade and morphological analysis showed that this strain should be described as a new Trebouxia species (in preparation). Another experimental approach was performed in order to explore how many algal taxa are compatible photobionts for this mycobiont. We combined P. muralis with diverse algal taxa, these were the following: Trebouxia arboricola, T. asymmetrica, T. decolorans, T. gigantea, T. higginsiae, T. impressa, T. incrustata, T. jamesii, T. "muralis", T. usneae, Asterochloris sp. For these resynthesis experiments we used different nutrient media.

Mycobionts were isolated both from thallus fragments and spores and cultured on different media. It was very important to find the optimal conditions for the in vitro growth of the mycobiont in order to produce higher quantities of biomass for identification and potential pharmaceutical or agricultural use of secondary metabolites. The best growth of the mycobiont was observed on G-LBM, MS and PDA media. The subcultures obtained differed with the pigmentation, especially when cultured on MS and G-LBM media the mycobiont produced a high quantity of unknown green pigment that was observed on the hyphae and even on the medium around the mycelium.

In order to check the influence of the photobiont on the production of secondary metabolites, we analysed specimens of P. muralis collected from different substrata and containing different Trebouxia species as photobionts. Using HPLC analysis we found that mainly usnic acid is produced and is some cases also small traces of unidentified substances, but no particular difference between thalli containing diverse photobionts was found.

We also analysed the impact of composition of media on the production of secondary metabolites. We compared chemical profiles of the mycobiont cultured on different media. It was found that the production of secondary metabolites is strongly dependant on the medium used, e.g.wWhen growing on modified BBM medium containing glucose the mycobiont did not produce any detectable substances; however mycobionts cultured on PDA, MS or G-LBM produced a wide range of secondary metabolites. Unfortunately, these are unknown substances that might be intermediates in the biosynthesis of usnic acid or the products of its hydrolysis, but it's also possible that a secondary biosynthetic pathway is activated and new substances are produced. In a future project it is planned to identify major substances produced by in-vitro cultured mycobionts.

In the next step we isolated microsatellite markers that could be used for population genetic studies of P. muralis. In the microsatellites isolation procedure we used pure mycobiont cultures in order to avoid algal contaminations. Among the clones sequenced 62 contained repeats. However, not all were unique- some of them were identical or chimeric sequences that decreased the number of positive clones. Finally 38 primer pairs were designed, among them 9 primer pairs were found to amplify polymorphic microsatellite loci from different specimens of P. muralis and these will be used for further population genetics studies. Additionally UV-light stress experiments were performed in order to test if UV-light stress may induce the production of additional metabolites or decrease the synthesis of the others.