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

The bacterial community structure of ecto-mycorrhizospheres on beech (Fagus sylvatica) grown in natural forest soil in southern Germany was examined b y fluorescence in situ hybridization (FISH) using fluorescent oligonucleoti de probes, targeting phylogenetic relevant sequences of the 16S and 23S rRN A. Lactarius subdulcis, L. vellereus, L. rubrocinctus and Laccaria amethyst ina were found to be the prevalent fungi forming ectomycorrhizae with F. sy lvatica. For FISH studies using confocal laser scanning microscopy, oligonu cleotide probes labeled with carboxymethylindocyanine-succinimidyl ester al lowed detection of associated bacteria, because the autofluorescence of ect omycorrhiza samples could be overcome in the infrared. Bacteria of the alph a-, beta and gamma-subclasses of the proteobacteria were detected in high n umbers on mantle surfaces, while members of other phylogenetically defined groups were found in smaller numbers. This contrasts with previous publishe d results on the cultivation of mycorrhiza-associated bacteria. Hybridizing bacteria were also found within damaged cells of the hyphal mantle of L. r ubrocinctus, as well as on emanating hyphae of L. amethystina. Using a newl y 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 alpha- and beta-subclasses of t he proteobacteria. The proportion of hybridizing bacteria varied between th e two ectomycorrhizae, which were thus host to distinct populations of bact eria.