Calcofluor white, an Alternative to Propidium Iodide for Plant Tissues Staining in Studies of Root Colonization by Fluorescent-tagged Rhizobia

Abstract

Aims: To study multiple bacterial colonization In vitro, several limitations are obvious. One of these limitations is the plant autofluorescence generally between green and red fluorescence depending on the plant sections. The most important limitation is the bacterial fluorescence labelling, compromised by different kind of variables. Here we report the use of a secure stain, Calcofluor White, in rhizobial and other kind of beneficial bacteria colonization studies. Study Design: Root colonization assays were designed to confirm the stability of Calcofluor White stain (Sigmaâ) in root cell walls. Place and Duration of Study: Every assay developed in this method article was performed using the technical resources at the Department of Microbiology and Genetics in the University of Salamanca (Spain) in 2013. Methodology: We have labelled rhizobia with two different kinds of fluorescent protein genes (gfp and rfp). We have co-inoculated Lactuca sativa and Daucus carota seedlings with two rhizobia: GFP-tagged Rhizobium sp. PEPV16 and RFP-tagged Mesorhizobium sp. CSLC01. Colonization assays were perfomed in several days post-inoculation, staining inoculated lettuce and carrot roots with Calcofluor White stain (Sigma®). Samples were monitorized for several days, using a fluorescence microscope (NIKON Eclipse 80i). Results: Bacterial attachment to plant tissues is observed by fluorescence microscopy after their labelling with fluorescent proteins. Our results show how Calcofluor White staining for plant tissues improves bacterial visualization in contrast with tissues stained with propidium iodide, a carcinogenic agent that cannot be used when bacteria are tagged with red fluorescent proteins such as RFP or mCherry. Conclusion: Calcofluor white is a non-carcinogenic and low toxic compound that has been classically used to stain fungi and plant tissues for different uses. Due to its low wavelength, calcofluor white may be used in combination with several fluorophores. In the present work we showed that this compound is a reliable alternative to propidium iodide for plant tissues staining in multiple rhizobial/bacterial colonization studies.