Abstract: Catalyzed reporter deposition fluorescent in situ hybridization（CARD⁃FISH）is an advanced technology to identify，track，and visualize root⁃associated microorganisms. It can accurately locate specific microbes in plant tissues，help us monitor the presence of microorganisms and their colonization strategy in the root system，and it is a powerful tool for studying root⁃associated microorganisms. With the advancement of molecular microbial ecological techniques，the combination of CARD⁃FISH and other approaches can efficiently analyze the functional properties of microorganisms in complex habitats. For example，the combination of CARD⁃FISH and nanometer secondary ion mass spectrometry（nanoSIMS）can simultaneously characterize the metabolic function and the phylogenetic properties of microorganism at the single⁃cell level. This article reviewed the principles，steps，development and combined technique of CARD⁃FISH，in order to provide a basis and reference for CARD⁃FISH to be used in the localization and functional analysis of plant tissue microorganisms.

Key words: CARD?FISH, plant?microbe interaction, root system, colonization, visualization