Bacterial community structure and colonization patterns of Fagus sylvatica L. ectomycorrhizospheres as determined by fluorescence in situ hybridization and confocal laser scanning microscopy

Abstract

The bacterial community structure of ecto- mycorrhizospheres on beech (Fagus sylvatica) grown in natural forest soil in southern Germany was exam- ined by fluorescence in situ hybridization (FISH) using fluorescent oligonucleotide probes, targeting phyloge- netic relevant sequences of the 16S and 23S rRNA. Lactarius subdulcis, L. vellereus, L. rubrocinctus and Laccaria amethystina were found to be the prevalent fungi forming ectomycorrhizae with F. sylvatica. For FISH studies using confocal laser scanning microsco- py, oligonucleotide probes labeled with carboxymethy- lindocyanine-succinimidyl ester allowed detection of associated bacteria, because the autofluorescence of ectomycorrhiza samples could be overcome in the infrared. Bacteria of the a-, b and g-subclasses of the proteobacteria were detected in high numbers on mantle surfaces, while members of other phylogeneti- cally defined groups were found in smaller numbers. This contrasts with previous published results on the cultivation of mycorrhiza-associated bacteria. Hybrid- izing bacteria were also found within damaged cells of the hyphal mantle of L. rubrocinctus, as well as on emanating hyphae of L. amethystina. Using a newly developed extraction protocol for bacteria associated with ectomycorhizas, the two most common fungi on F. sylvatica, L. vellereus and L. subdulcis, were mostly associated with members of the a- and b-subclasses of the proteobacteria. The proportion of hybridizing bac- teria varied between the two ectomycorrhizae, which were thus host to distinct populations of bacteria.