Label-Free Functional Analysis of Root-Associated Microbes with Dynamic Quantitative Oblique Back-illumination Microscopy

• Type of Material: Article
• Authors: Caroline Filan, Georgia Institute of Technology; Madison Green, Georgia Institute of Technology; Abigail Diering, Georgia Institute of Technology; Marcus T. Cicerone, Georgia Institute of Technology; Lily S. Cheung, Georgia Institute of Technology; Joel E. Kostka, Georgia Institute of Technology; Francisco E. Robles, Emory University
• Language: English
• Date: 2023-11-02
• Publisher: NIH
• Publication Version: Preprint (Prior to Peer Review)
• Copyright Statement: 2023 Research Square
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): https://doi.org/10.21203/rs.3.rs-3517586/v1
• Title of Journal or Parent Work: Research Square
• Start Page: 3517586
• Grant/Funding Information: This work was funded by the Department of Energy Biological and Environmental research (DOE BER DE-SC0022121), the National Institute of Health National Institute of General Medical Sciences (NIH NIGMS R35GM147437), and the National Science Foundation Graduate Research Fellowship (NSF GRFP DGE-2039655).
• Keywords: Rahnella aquatilis; Sinorhizobium meliloti; dynamic quantitative oblique back-illumination microscopy (DqOBM); Sustainable Agriculture; zotobacter vinelandii
• Research Categories: Environmental Sciences; Biology, Ecology

Abstract

The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the dynamic activity of diazotrophs in vitro and in situ. Our experiments involved three different diazotrophs (Sinorhizobium meliloti, Azotobacter vinelandii, and Rahnella aquatilis) cultured on media with amendments of carbon and nitrogen sources. Over five days, we observed increased dynamic activity in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize bacterial activity within the root cap and elongation zone of Arabidopsis thaliana primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial activity and provide insights into plant-microbe interactions in situ, offering a valuable tool for advancing our understanding of sustainable agriculture.