Poster: Diseases of Plants: Disease Detection & Diagnosis
519-P
Optimization of a fluorescence in situ hybridization assay for detection and visualization of Plasmopara obducens from plants and soil
C. SALGADO-SALAZAR (1) (1) USDA-ARS, Rutgers University, U.S.A.
Plasmopara obducens is the biotrophic oomycete responsible for downy mildew of ornamental impatiens, a destructive disease that significantly impacts the production of Impatiens walleriana in the United States. To date, there are no methods to detect P. obducens from asymptomatic plants, which may be contributing to the spread of the disease. Furthermore, the extent that P. obducens uses oospores to colonize and persist in the soil is undetermined. 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. A FISH assay for the impatiens downy mildew pathogen was designed by preparing DNA sequence alignments of the rDNA-ITS region of P. obducens and other related taxa to identify suitable regions for species-specific probe development. Because P. obducens cannot be propagated in vitro, we developed a custom E. coli expression vector that transcribes the rDNA-ITS gene sequence of P. obducens for use as a control and to allow for standardized fluorescence signal quantification (clone-FISH). The optimized probe hybridization stringency resulted in good fluorescence signal compared to those of the negative controls. The standardized protocol developed using the clone-FISH reagents will be used in subsequent experiments to directly visualize P. obducens in situ from plant and soil samples