Leaf-FISH: In Situ Hybridization Method for Visualizing Bacterial Taxa on Plant Surfaces

Area of Science:

• Microbial ecology
• Plant pathology
• Microscopy

Background:

• Accurate spatial characterization of phyllosphere microbial communities is essential for understanding microbe-microbe and microbe-plant interactions.
• Leaf surfaces present imaging challenges due to high autofluorescence, hindering visualization of microbial communities.
• Existing methods struggle to provide high-resolution, spatially resolved data of bacteria within the phyllosphere.

Purpose of the Study:

• To introduce and describe the Leaf-FISH method for high-resolution spatial characterization of microbial communities on leaf surfaces.
• To overcome the challenge of leaf autofluorescence for improved visualization of bacteria in the phyllosphere.
• To enable simultaneous identification of multiple bacterial taxa within a structural context.

Main Methods:

• Leaf-FISH employs specific leaf pretreatments to mitigate autofluorescence.
• Spectral imaging confocal microscopy is utilized for high-resolution visualization.
• Image post-processing reconstructs a structural context from residual autofluorescence.
• Combinatorial labeling with multiple taxon-specific probes allows simultaneous identification of diverse bacterial taxa.

Main Results:

• Leaf-FISH successfully visualizes bacterial taxa on leaf surfaces within a structural context.
• The method effectively overcomes challenges posed by leaf autofluorescence.
• Simultaneous identification of multiple bacterial taxa is achievable using this technique.
• High-resolution spatial characterization of phyllosphere microbial communities is enabled.

Conclusions:

• Leaf-FISH is a robust method for the spatial characterization of phyllosphere bacteria.
• This technique enhances the understanding of microbe-microbe and microbe-plant interactions.
• Leaf-FISH provides a valuable tool for microbial ecology and plant pathology research.