Dataset Information

Leaf-FISH: Microscale Imaging of Bacterial Taxa on Phyllosphere.

ABSTRACT: Molecular methods for microbial community characterization have uncovered environmental and plant-associated factors shaping phyllosphere communities. Variables undetectable using bulk methods can play an important role in shaping plant-microbe interactions. Microscale analysis of bacterial dynamics in the phyllosphere requires imaging techniques specially adapted to the high autoflouresence and 3-D structure of the leaf surface. We present an easily-transferable method (Leaf-FISH) to generate high-resolution tridimensional images of leaf surfaces that allows simultaneous visualization of multiple bacterial taxa in a structurally informed context, using taxon-specific fluorescently labeled oligonucleotide probes. Using a combination of leaf pretreatments coupled with spectral imaging confocal microscopy, we demonstrate the successful imaging bacterial taxa at the genus level on cuticular and subcuticular leaf areas. Our results confirm that different bacterial species, including closely related isolates, colonize distinct microhabitats in the leaf. We demonstrate that highly related Methylobacterium species have distinct colonization patterns that could not be predicted by shared physiological traits, such as carbon source requirements or phytohormone production. High-resolution characterization of microbial colonization patterns is critical for an accurate understanding of microbe-microbe and microbe-plant interactions, and for the development of foliar bacteria as plant-protective agents.

SUBMITTER: Peredo EL

PROVIDER: S-EPMC5767230 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Leaf-FISH: Microscale Imaging of Bacterial Taxa on Phyllosphere.

Peredo Elena L EL Simmons Sheri L SL

Frontiers in microbiology 20180109

Molecular methods for microbial community characterization have uncovered environmental and plant-associated factors shaping phyllosphere communities. Variables undetectable using bulk methods can play an important role in shaping plant-microbe interactions. Microscale analysis of bacterial dynamics in the phyllosphere requires imaging techniques specially adapted to the high autoflouresence and 3-D structure of the leaf surface. We present an easily-transferable method (Leaf-FISH) to generate h ...[more]

PMID: 29375531

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Project description:Every year, deciduous trees shed their leaves, and when new leaves emerge next spring, they establish a characteristic bacterial leaf community. In this exploratory study, we assessed the bacterial phyllosphere (aboveground plant surfaces) of eight London plane trees (Platanus × acerifolia) in Antwerp and Milan by sampling weekly during leaf emergence and expansion. We sampled the surfaces of different tree compartments: leaves, leaf buds, branches, and trunk, for up to 6 weeks. Phyllosphere community composition was most strongly determined by tree compartment. Only the communities on the emerging leaves showed changing dynamics over time. The rate of change in the leaf phyllosphere composition, expressed as the beta dissimilarity between consecutive time points, was very high following leaf emergence, with decreasing speed over time, indicating that these communities stabilize over time. We also identified cooccurring groups of bacteria associated with potential stages of ecological succession on the leaves and accordingly named them general cluster, early cluster, middle cluster, and late cluster. Taxa of the general cluster were not only more abundant than the others on leaves, but they were also widespread on other tree compartments. The late cluster was most pronounced in trees surrounded by trafficked urban land use. This study mainly generates hypotheses on the ecological succession on the emerging leaves of deciduous trees in urban environments and contributes to understanding the development of the tree leaf phyllosphere in spring. IMPORTANCE Improving our understanding of phyllosphere ecology is key in successfully applying bacterial biological agents or modulating the leaf microbiome in order to achieve valuable ecosystem services, such as plant protection, plant growth, air purification, and developing a healthy human immune system. Modulation of the phyllosphere microbiome in the field works only with variable success. To improve the impact of our applications in the field, a better understanding of the ecological principles governing phyllosphere dynamics is required. This exploratory study demonstrates how the combination of different analyses of a chronosequence of bacterial communities can provide new ecological insights. With a limited number of sampled trees, we demonstrated different indications of ecological succession of bacterial communities in the leaves and observed a potential impact of intensely trafficked land use becoming apparent in the leaf bacterial communities approximately 3 weeks after leaf emergence, consisting of a separate stage in community development.

Project description:Artificial surfaces are routinely used instead of leaves to enable a reductionist approach in phyllosphere microbiology, the study of microorganisms residing on plant leaf surfaces. Commonly used artificial surfaces include, flat surfaces, such as metal and nutrient agar, and microstructured surfaces, such as isolate leaf cuticles or reconstituted leaf waxes. However, interest in replica leaf surfaces as an artificial surface is growing, as replica surfaces provide an improved representation of the complex topography of leaf surfaces. To date, leaf surfaces have predominantly been replicated for their superhydrophobic properties. In contrast, in this paper we investigated the potential of agarose, the elastomer polydimethylsiloxane (PDMS), and gelatin as replica leaf surface materials for phyllosphere microbiology studies. Using a test pattern of pillars, we investigated the ability to replicate microstructures into the materials, as well as the degradation characteristics of the materials in environmental conditions. Pillars produced in PDMS were measured to be within 10% of the mold master and remained stable throughout the degradation experiments. In agarose and gelatin the pillars deviated by more than 10% and degraded considerably within 48 hours in environmental conditions. Furthermore, we investigated the surface energy of the materials, an important property of a leaf surface, which influences resource availability and microorganism attachment. We found that the surface energy and bacterial viability on PDMS was comparable to isolated Citrus × aurantium and Populus × canescens leaf cuticles. Hence indicating that PDMS is the most suitable material for replica leaf surfaces. In summary, our experiments highlight the importance of considering the inherent material properties when selecting a replica leaf surface for phyllosphere microbiology studies. As demonstrated, a PDMS replica leaf offers a control surface that can be used for investigating microbe-microbe and microbe-plant interactions in the phyllosphere, which will enable mitigation strategies against pathogens to be developed.

Dataset Information

Leaf-FISH: Microscale Imaging of Bacterial Taxa on Phyllosphere.

Publications

Leaf-FISH: Microscale Imaging of Bacterial Taxa on Phyllosphere.

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