Spatial distribution analyses of natural phyllosphere-colonizing bacteria on Arabidopsis thaliana revealed by fluorescence in situ hybridization.

Bacterial colonizers of the aerial parts of plants, or phyllosphere, have been identified on a number of different plants using cultivation-dependent and independent methods. However, the spatial distribution at the micrometer scale of different main phylogenetic lineages is not well documented and mostly based on fluorescence-tagged model strains. In this study, we developed and applied a spatial explicit approach that allowed the use of fluorescence in situ hybridization (FISH) to study bacterial phylloplane communities of environmentally grown Arabidopsis thaliana. We found on average 5.4 × 10(6) bacteria cm(-2) leaf surface and 1.5 × 10(8) bacteria g(-1) fresh weight. Furthermore, we found that the total biomass in the phylloplane was normally distributed. About 31% of the bacteria found in the phylloplane did not hybridize to FISH probes but exhibited infrared autofluorescence indicative for aerobic anoxygenic phototrophs. Four sets of FISH probes targeting Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes were sufficient to identify all other major contributors of the phylloplane community based on general bacterial probing. Spatial aggregation patterns were observed for all probe-targeted populations at distances up to 7 μm, with stronger tendencies to co-aggregate for members of the same phylogenetic group. Our findings contribute to a bottom-up description of leaf surface community composition.