Localization of bacteria in lichens from Alpine soil crusts by fluorescence in situ hybridization

• Localization of bacteria in lichen–soil interface was analyzed by FISH and CLSM.
• Six species of Alpine soil crust lichens were selected.
• An improved FISH protocol is presented for soil crust material.
• Specific FISH probes were applied to detect the major bacterial groups.
• The proposed protocol enables to study other ecologically important soil crust habitats.

Lichens are prominent components of many biological soil crusts. Owing to their persistence, lichen thalli create microhabitats for other microbes. Here, the structure of bacterial communities at the thallus–soil interface in lichen soil crusts was studied by using fluorescence in situ hybridization (FISH), confocal laser scanning microscopy (CLSM) and 3D image reconstruction. Terricolous lichen thalli above the tree-line in open habitats of the Austrian Alps were sampled. We selected six lichen species associated with green algal photobionts: Arthrorhaphis citrinella, Baeomyces placophyllus, B. rufus, Icmadophila ericetorum, Psora decipiens and Trapeliopsis granulosa. Alphaproteobacteria and Acidobacteria are predominant in these soil crust lichens, where the latter are frequently present in the lower part of lichen thalli and in the hypothallosphere. In the inconspicuous thallus structures of Arthrorhaphis citrinella, Baeomyces rufus, Icmadophila ericetorum and Trapeliopsis granulosa we observed association of bacteria with algal cells in soil particles and on the outer surface of the mycobiont–photobiont aggregates. We found bacterial cells intermixed with photobiont cells in the lower part of the lichen thalli and as small colonies on the surface of the squamules of Baeomyces placophyllus and Psora decipiens. Moreover, technical issues of performing FISH and confocal microscopy with biological soil crusts are discussed.