Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH).

Background: is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of that causes high crop loss. Currently, there are no available methods for the microscopic detection of from leaves of impatiens, which may be contributing to the spread of the disease. Fluorescence in situ hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide probes to detect target organisms from the environment. To study this important pathogen, we developed and standardized a FISH technique for the visualization of from tissues using a species-specific 24-mer oligonucleotide probe designed to target a region of the rRNA internal transcribed spacer 2 (ITS2).

Results: Since cannot be propagated in vitro, we developed a custom expression vector that transcribes the rRNA-ITS target sequence (clone-FISH) for use as a control and to optimize hybridization conditions. The FISH assay could detect sporangiophores, sporangia and oospores, and hyphae from naturally infected leaves and stems. Cross-reactivity was not observed from plant tissue, and the assay did not react when applied to with self-ligated plasmids and non-target oomycete species.

Conclusions: This FISH protocol may provide a valuable tool for the study of this disease and could potentially be used to improve early monitoring of , substantially reducing the persistence and spread of this destructive plant pathogen.