Background Elucidating the spatial structure of host-associated microbial communities is essential for understanding taxon-taxon interactions within the microbiota and between microbiota and host. Macroalgae are colonized by complex microbial communities, suggesting intimate symbioses that likely play key roles in both macroalgal and bacterial biology, yet little is known about the spatial organization of microbes associated with macroalgae. Canopy-forming kelp are ecologically significant, fixing teragrams of carbon per year in coastal kelp forest ecosystems. We characterized the micron-scale spatial organization of bacterial communities on blades of the kelp Nereocystis luetkeana using fluorescence in situ hybridization and spectral imaging with a probe set combining phylum-, class-, and genus-level probes to localize and identify > 90% of the microbial community. Results We show that kelp blades host a dense microbial biofilm composed of disparate microbial taxa in close contact with one another. The biofilm is spatially differentiated, with clustered cells of the dominant symbiont Granulosicoccus sp. (Gammaproteobacteria) close to the kelp surface and filamentous Bacteroidetes and Alphaproteobacteria relatively more abundant near the biofilm-seawater interface. A community rich in Bacteroidetes colonized the interior of kelp tissues. Microbial cell density increased markedly along the length of the kelp blade, from sparse microbial colonization of newly produced tissues at the meristematic base of the blade to an abundant microbial biofilm on older tissues at the blade tip. Kelp from a declining population hosted fewer microbial cells compared to kelp from a stable population. Conclusions Imaging revealed close association, at micrometer scales, of different microbial taxa with one another and with the host. This spatial organization creates the conditions necessary for metabolic exchange among microbes and between host and microbiota, such as provisioning of organic carbon to the microbiota and impacts of microbial nitrogen metabolisms on host kelp. The biofilm coating the surface of the kelp blade is well-positioned to mediate interactions between the host and surrounding organisms and to modulate the chemistry of the surrounding water column. The high density of microbial cells on kelp blades (105–107 cells/cm2), combined with the immense surface area of kelp forests, indicates that biogeochemical functions of the kelp microbiome may play an important role in coastal ecosystems.

15Macroalgae are colonized by complex and diverse microbial communities that are distinct from 16 those on inert substrates, suggesting intimate symbioses that likely play key roles in both 17 macroalgal and bacterial biology. Canopy-forming kelp fix teragrams of carbon per year in 18 coastal kelp forest ecosystems, yet little is known about the structure and development of their 19 associated microbial communities. We characterized the spatial organization of bacterial 20 communities on blades of the canopy-forming kelp Nereocystis luetkeana using fluorescence in 21 situ hybridization and spectral imaging with a probe set combining phylum, class and genus-22Importance 35 The microbial community coating the surfaces of macroalgae may play a key but underexplored 36 role both in the biology of the macroalgal host and in the biogeochemistry of the coastal ocean. 37We show that photosynthetic blades of the canopy-forming kelp Nereocystis luetkeana host a 38 complex microbial biofilm that is both dense and spatially differentiated. Microbes of different 39 taxa are in intimate cell-to-cell contact with one another; microbial cells invade the interior of 40 kelp cells as well as cover their external surfaces; and a subset of the surface microbiota projects 41 into the water column. These results highlight the potential for metabolic interactions between 42 key members of the kelp microbiome as well as between microbes and their host. The dense 43 layer of microbes coating the surface of the kelp blade is well-positioned to mediate interactions 44 between the host and surrounding organisms and to modulate the chemistry of the surrounding 45 water column. 46 the microbiome and condition of habitat-forming kelp.