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Title (Primary) Microbial identification, high-resolution microscopy and spectrometry of the rhizosphere in its native spatial context
Author Bandara, C.D.; Schmidt, M.; Davoudpour, Y.; Stryhanyuk, H.; Richnow, H.H.; Musat, N.
Journal Frontiers in Plant Science
Year 2021
Department ISOBIO
Volume 12
Page From art. 668929
Language englisch
Topic T8 Georesources
Data links https://doi.org/10.48758/ufz.11158
Supplements https://www.frontiersin.org/articles/10.3389/fpls.2021.668929/full#supplementary-material
Keywords CARD-FISH, Chemical microscopy, HIM, Nano-SIMS, Resin embedding, rhizosphere, soil bacteria, spatial distribution, Water-jet cutting
UFZ wide themes ProVIS;
Abstract During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e. mapping of elemental, isotopic and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro- to nano-meter spatial resolution. Our approach includes i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (ยต-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance.
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=24272
Bandara, C.D., Schmidt, M., Davoudpour, Y., Stryhanyuk, H., Richnow, H.H., Musat, N. (2021):
Microbial identification, high-resolution microscopy and spectrometry of the rhizosphere in its native spatial context
Front. Plant Sci. 12 , art. 668929