Corrigendum: Microbial identification, high-resolution microscopy and spectrometry of the rhizosphere in its native spatial context.

[This corrects the article DOI: 10.3389/fpls.2021.668929.].

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context.

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.

Resolving Bio-Nano Interactions of E. coli Bacteria-Dragonfly Wing Interface with Helium Ion and 3D-Structured Illumination Microscopy to Understand Bacterial Death on Nanotopography.

Obtaining a comprehensive understanding of the bactericidal mechanisms of natural nanotextured surfaces is crucial for the development of fabricated nanotextured surfaces with efficient bactericidal activity. However, the scale, nature, and speed of bacteria-nanotextured surface interactions make the characterization of the interaction a challenging task. There are currently several different opinions regarding the possible mechanisms by which bacterial membrane damage occurs upon interacting with nanotextured surfaces. Advanced imaging methods could clarify this by enabling visualization of the interaction. Charged particle microscopes can achieve the required nanoscale resolution but are limited to dry samples. In contrast, light-based methods enable the characterization of living (hydrated) samples but are limited by the resolution achievable. Here we utilized both helium ion microscopy (HIM) and 3D structured illumination microscopy (3D-SIM) techniques to understand the interaction of Gram-negative bacterial membranes with nanopillars such as those found on dragonfly wings. Helium ion microscopy enables cutting and imaging at nanoscale resolution, while 3D-SIM is a super-resolution optical microscopy technique that allows visualization of live, unfixed bacteria at ∼100 nm resolution. Upon bacteria-nanopillar interaction, the energy stored due to the bending of natural nanopillars was estimated and compared with fabricated vertically aligned carbon nanotubes. With the same deflection, shorter dragonfly wing nanopillars store slightly higher energy compared to carbon nanotubes. This indicates that fabricated surfaces may achieve similar bactericidal efficiency as dragonfly wings. This study reports in situ characterization of bacteria-nanopillar interactions in real-time close to its natural state. These microscopic approaches will help further understanding of bacterial membrane interactions with nanotextured surfaces and the bactericidal mechanisms of nanotopographies so that more efficient bactericidal nanotextured surfaces can be designed and fabricated, and their bacteria-nanotopography interactions can be assessed in situ.

Effect of Precursor on Antifouling Efficacy of Vertically-Oriented Graphene Nanosheets.

Antifouling efficacy of graphene nanowalls, i.e., substrate-bound vertically-oriented graphene nanosheets, has been demonstrated against biofilm-forming Gram-positive and Gram-negative bacteria. Where graphene nanowalls are typically prepared using costly high-temperature synthesis from high-purity carbon precursors, large-scale applications demand efficient, low-cost processes. The advancement of plasma enabled synthesis techniques in the production of nanomaterials has opened a novel and effective method for converting low-cost natural waste resources to produce nanomaterials with a wide range of applications. Through this work, we report the rapid reforming of sugarcane bagasse, a low-value by-product from sugarcane industry, into high-quality vertically-oriented graphene nanosheets at a relatively low temperature of 400 °C. Electron microscopy showed that graphene nanowalls fabricated from methane were significantly more effective at preventing surface attachment of Gram-negative rod-shaped Escherichia coli compared to bagasse-derived graphene, with both surfaces showing antifouling efficacy comparable to copper. Attachment of Gram-positive coccal Staphylococcus aureus was lower on the surfaces of both types of graphene compared to that on copper, with bagasse-derived graphene being particularly effective. Toxicity to planktonic bacteria estimated as a reduction in colony-forming units as a result of sample exposure showed that both graphenes effectively retarded cell replication.

Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia coli.

Nanotextured surfaces (NTSs) are critical to organisms as self-adaptation and survival tools. These NTSs have been actively mimicked in the process of developing bactericidal surfaces for diverse biomedical and hygiene applications. To design and fabricate bactericidal topographies effectively for various applications, understanding the bactericidal mechanism of NTS in nature is essential. The current mechanistic explanations on natural bactericidal activity of nanopillars have not utilized recent advances in microscopy to study the natural interaction. This research reveals the natural bactericidal interaction between E. coli and a dragonfly wing's (Orthetrum villosovittatum) NTS using advanced microscopy techniques and proposes a model. Contrary to the existing mechanistic models, this experimental approach demonstrated that the NTS of Orthetrum villosovittatum dragonfly wings has two prominent nanopillar populations and the resolved interface shows membrane damage occurred without direct contact of the bacterial cell membrane with the nanopillars. We propose that the bacterial membrane damage is initiated by a combination of strong adhesion between nanopillars and bacterium EPS layer as well as shear force when immobilized bacterium attempts to move on the NTS. These findings could help guide the design of novel biomimetic nanomaterials by maximizing the synergies between biochemical and mechanical bactericidal effects.