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 (μ-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.
Highlights
Soils are heterogeneous mixtures of various organic materials, mineral particles, and pores, which provide a matrix for plant growth and resource recycling by microbiome
Several studies show that the choice of fixative and their time of action can affect the overall chemistry of the sample (Pathan et al, 2008, 2010; Musat et al, 2016; Fiedler et al, 2018)
Our results demonstrate Helium Ion Microscopy (HIM) is a well-suited for non-quantitative imaging of the embedded rhizosphere sample to prepare treasure maps of the interested region of interest (RoI) and image registration
Summary
Soils are heterogeneous mixtures of various organic materials, mineral particles, and pores, which provide a matrix for plant growth and resource recycling by microbiome. The structural and biochemical complexity and spatial arrangement of microbes of the rhizosphere as the main driver of terrestrial biogeochemical cycles is little understood To explore such relationships between multiple components (Ganther et al, 2020), it is crucial to characterize all soil components whilst keeping their structural and chemical integrity i.e., chemical composition and spatial organization of minerals, non-living organic matter and microbial counterparts such as bacteria, fungi, and plant roots during the analysis (Nunan et al, 2007; Mueller et al, 2013, 2019; Steffens et al, 2017; Vidal et al, 2018; Juyal et al, 2019, 2020; Fadel et al, 2020). A comprehensive chemical characterization coupled to microbial identification and distribution at spatial scales relevant to biogeochemical processes is necessary to achieve a systematic understanding of the key factors governing the self-organization of the rhizosphere (Nunan et al, 2007; Baveye et al, 2018; Juyal et al, 2019)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
