The phyllosphere – the aerial parts of plants – is an important microbial habitat that is home to diverse microbial communities. The spatial organization of bacterial cells on leaf surfaces is non-random, and correlates with leaf microscopic features. Yet, the role of microscale interactions between bacterial cells therein is not well understood. Here, we ask how interactions between immigrant bacteria and resident microbiota affect the spatial organization of the combined community. By means of live imaging in a simplified in vitro system, we studied the spatial organization, at the micrometer scale, of the biocontrol agent Pseudomonas fluorescens A506 and the plant pathogen P. syringae B728a when introduced to pear and bean leaf microbiota (the corresponding native plants of these strains). We found significant co-localization of immigrant and resident microbial cells at distances of a few micrometers, for both strains. Interestingly, this co-localization was in part due to preferential attachment of microbiota cells near newly formed P. fluorescens aggregates. Our results indicate that two-way immigrant bacteria – resident microbiota interactions affect the microscale spatial organization of leaf microbiota, and possibly that of other surface-related microbial communities.
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Lindow SE, Brandl MT. Microbiology of the phyllosphere. Appl Environ Microbiol. 2003;69:1875–83.
Lindow SE, Leveau JH. Phyllosphere microbiology. Curr Opin Biotechnol. 2002;13:238–43.
Vorholt JA. Microbial life in the phyllosphere. Nat Rev Microbiol. 2012;10:828.
Vacher C, Hampe A, Porté AJ, Sauer U, Compant S, Morris CE. The phyllosphere: microbial jungle at the plant–climate interface. Annu Rev Ecol Evol Syst. 2016;47:1–24.
Bringel F, Couée I. Pivotal roles of phyllosphere microorganisms at the interface between plant functioning and atmospheric trace gas dynamics. Front Microbiol. 2015;6:486.
Redford AJ, Bowers RM, Knight R, Linhart Y, Fierer N. The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves. Environ Microbiol. 2010;12:2885–93.
Rastogi G, Sbodio A, Tech JJ, Suslow TV, Coaker GL, Leveau JH. Leaf microbiota in an agroecosystem: spatiotemporal variation in bacterial community composition on field-grown lettuce. ISME J. 2012;6:1812.
Beattie GA, Lindow SE. Bacterial colonization of leaves: a spectrum of strategies. Phytopathology. 1999;89:353–359.
Agler MT, Ruhe J, Kroll S, Morhenn C, Kim S-T, Weigel D, et al. Microbial hub taxa link host and abiotic factors to plant microbiome variation. PLoS Biol. 2016;14:e1002352.
Laforest-Lapointe I, Messier C, Kembel SW. Tree phyllosphere bacterial communities: exploring the magnitude of intra-and inter-individual variation among host species. PeerJ. 2016;4:e2367.
Monier J-M, Lindow S. Frequency, size, and localization of bacterial aggregates on bean leaf surfaces. Appl Environ Microbiol. 2004;70:346–55.
Tecon R, Leveau JH. The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology. Environ Microbiol. 2012;14:1325–32.
Remus-Emsermann MN, Lücker S, Müller DB, Potthoff E, Daims H, Vorholt JA. Spatial distribution analyses of natural phyllosphere-colonizing bacteria on A rabidopsis thaliana revealed by fluorescence in situ hybridization. Environ Microbiol. 2014;16:2329–40.
Morris CE, Monier J, Jacques M. Methods for observing microbial biofilms directly on leaf surfaces and recovering them for isolation of culturable microorganisms. Appl Environ Microbiol. 1997;63:1570–6.
Esser DS, Leveau JH, Meyer KM, Wiegand K. Spatial scales of interactions among bacteria and between bacteria and the leaf surface. FEMS Microbiol Ecol. 2015;91:fiu034.
Remus-Emsermann MN, Schlechter RO. Phyllosphere microbiology: at the interface between microbial individuals and the plant host. N. Phytologist. 2018;218:1327–33.
Monier J-M, Lindow S. Spatial organization of dual-species bacterial aggregates on leaf surfaces. Appl Environ Microbiol. 2005;71:5484–93.
Peredo EL, Simmons SL. Leaf-FISH: microscale imaging of bacterial taxa on phyllosphere. Front Microbiol. 2018;8:2669.
Remus-Emsermann MN, Tecon R, Kowalchuk GA, Leveau JH. Variation in local carrying capacity and the individual fate of bacterial colonizers in the phyllosphere. ISME J. 2012;6:756.
Remus-Emsermann MN, Kowalchuk GA, Leveau JH. Single-cell versus population-level reproductive success of bacterial immigrants to pre-colonized leaf surfaces. Environ Microbiol Rep. 2013;5:387–92.
Monier J-M, Lindow S. Aggregates of resident bacteria facilitate survival of immigrant bacteria on leaf surfaces. Microb Ecol. 2005;49:343–52.
Poza-Carrion C, Suslow T, Lindow S. Resident bacteria on leaves enhance survival of immigrant cells of Salmonella enterica. Phytopathology. 2013;103:341–51.
Grinberg M, Orevi T, Kashtan N. Bacterial surface colonization, preferential attachment and fitness under periodic stress. PLoS Comput Biol. 2019;15:e1006815.
Beattie GA, Lindow SE. Comparison of the behavior of epiphytic fitness mutants of Pseudomonas syringae under controlled and field conditions. Appl Environ Microbiol. 1994;60:3799–808.
Loper JE, Lindow SE. Lack of evidence for the in situ fluorescent pigment production by Pseudomonas syringae pv. syringae on bean leaf surfaces. J Phytopathol. 1987;77:1449–54.
Wilson M, Hirano S, Lindow S. Location and survival of leaf-associated bacteria in relation to pathogenicity and potential for growth within the leaf. Appl Environ Microbiol. 1999;65:1435–43.
Stockwell V, Johnson K, Sugar D, Loper J. Control of fire blight by Pseudomonas fluorescens A506 and Pantoea vagans C9-1 applied as single strains and mixed inocula. Phytopathology. 2010;100:1330–9.
Wilson M, Lindow S. Interactions between the biological control agent Pseudomonas fluorescens A506 and Erwinia amylovora in pear blossoms. Phytopathology. 1993;83:117–23.
Choi K-H, Schweizer HP. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat Protoc. 2006;1:153.
Morris CE, Monier J-M, Jacques M-A. A technique to quantify the population size and composition of the biofilm component in communities of bacteria in the phyllosphere. Appl Environ Microbiol. 1998;64:4789–95.
Wang Q, Niemi J, Tan CM, You L, West M. Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy. Cytometry A. 2010;77:101–10.
Daims H, Lücker S, Wagner M. Daime, a novel image analysis program for microbial ecology and biofilm research. Environ Microbiol. 2006;8:200–13.
Daims H, Wagner M. In situ techniques and digital image analysis methods for quantifying spatial localization patterns of nitrifiers and other microorganisms in biofilm and flocs. Methods Enzymol. 2011;496:185–215.
Reed M, Howard C. Stereological estimation of covariance using linear dipole probes. J Microsc. 1999;195:96–103.
Van Der Wal A, Tecon R, Kreft J-U, Mooij WM, Leveau JH. Explaining bacterial dispersion on leaf surfaces with an individual-based model (PHYLLOSIM). PloS ONE. 2013;8:e75633.
Schmidt H, Nunan N, Höck A, Eickhorst T, Kaiser C, Woebken D, et al. Recognizing patterns: spatial analysis of observed microbial colonization on root surfaces. Front Environ Sci. 2018;6:61.
Gantner S, Schmid M, Dürr C, Schuhegger R, Steidle A, Hutzler P, et al. In situ quantitation of the spatial scale of calling distances and population density-independent N-acylhomoserine lactone-mediated communication by rhizobacteria colonized on plant roots. FEMS Microbiol Ecol. 2006;56:188–94.
Schillinger C, Petrich A, Lux R, Riep B, Kikhney J, Friedmann A, et al. Co-localized or randomly distributed? Pair cross correlation of in vivo grown subgingival biofilm bacteria quantified by digital image analysis. PLoS ONE. 2012;7:e37583.
Mercier J, Lindow S. Role of leaf surface sugars in colonization of plants by bacterial epiphytes. Appl Environ Microbiol. 2000;66:369–74.
Zhao K, Tseng BS, Beckerman B, Jin F, Gibiansky ML, Harrison JJ, et al. Psl trails guide exploration and microcolony formation in early P. aeruginosa biofilms. Nature. 2013;497:388.
Hödl I, Hödl J, Wörman A, Singer G, Besemer K, Battin TJ. Voronoi tessellation captures very early clustering of single primary cells as induced by interactions in nascent biofilms. PloS ONE. 2011;6:e26368.
Mittelviefhaus M, Müller DB, Zambelli T, Vorholt JA. A modular atomic force microscopy approach reveals a large range of hydrophobic adhesion forces among bacterial members of the leaf microbiota. ISME J. 2019;13:1878–82.
Laganenka L, Colin R, Sourjik V. Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli. Nat Commun. 2016;7:12984.
Laganenka L, Sourjik V. Autoinducer 2-dependent Escherichia coli biofilm formation is enhanced in a dual-species coculture. Appl Environ Microbiol. 2018;84:e02638–17.
Dulla G, Lindow SE. Quorum size of Pseudomonas syringae is small and dictated by water availability on the leaf surface. Proc Natl Acad Sci. 2008;105:3082–7.
Bertsche U, Mayer C, Götz F, Gust AA. Peptidoglycan perception—sensing bacteria by their common envelope structure. Int J Med Microbiol. 2015;305:217–23.
Monier J-M, Lindow S. Differential survival of solitary and aggregated bacterial cells promotes aggregate formation on leaf surfaces. Proc Natl Acad Sci. 2003;100:15977–82.
Grinberg M, Orevi T, Steinberg S, Kashtan N. Bacterial survival in microscopic surface wetness. eLife. 2019;8:e48508.
Shank EA, Kolter R. New developments in microbial interspecies signaling. Curr Opin Microbiol. 2009;12:205–14.
We thank Y. Hadar for valuable comments on the manuscript. We thank S. Lindow for kindly providing bacterial strains, and R. Feuchtwanger from Gan Hasadeh and N. Shachar for providing fresh leaves for this study. JP acknowledges the Lady Davis Trust for a postdoctoral fellowship. MB acknowledges the Rudin MSc scholarship. This work was supported by a research grant to NK from the James S. McDonnell Foundation (Studying Complex Systems Scholar Award, Grant #220020475) and from the Israel Science Foundation (ISF #1396/19).
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Steinberg, S., Grinberg, M., Beitelman, M. et al. Two-way microscale interactions between immigrant bacteria and plant leaf microbiota as revealed by live imaging. ISME J (2020). https://doi.org/10.1038/s41396-020-00767-z