Detection and visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH)

Authors: SALGADO-SALAZAR, CATALINA - Orise Fellow; Bauchan, Gary; WALLACE, EMMA - Orise Fellow; Crouch, Jo Anne

Submitted to: Plant Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/2018
Publication Date: 10/24/2018
Citation: Salgado-Salazar, C., Bauchan, G.R., Wallace, E.C., Crouch, J.A. 2018. Detection and visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH). Plant Methods. 14:92. https://doi.org/10.1186/s13007-018-0362-z.
DOI: https://doi.org/10.1186/s13007-018-0362-z

Interpretive Summary: Downy mildew is a deadly disease of impatiens that occurs when plants are infected by a type of water mold. When the disease is active, the presence of the downy mildew is very obvious, with white masses of the mold covering the lower leaf surface. But during early stages of the disease, the downy mildew mold is invisible to the naked eye. This allows the disease to be unknowingly spread around by infected plants that don't show any symptoms or signs of infection. In this study, a new method was developed to detect the presence of impatiens downy mildew from infected plants before disease symptoms are present. The method, which is based on a technique called fluorescent in situ hybridization--or FISH-- relies on the very specific signature of the downy mildew's DNA, producing a brightly colored fluorescent signal whenever the mildew is present. The FISH method is able to detect the downy mildew, even when it is only present inside the plant and is not found on the exterior. This research is significant because it will allow for early detection of impatiens downy mildew infections, before the plant becomes sick and spreads the disease. This research will be useful to growers, breeders, plant pathologists, extension personnel and quarantine officials who work to minimize the transmission and impact of impatiens downy mildew.

Technical Abstract: Plasmopara obducens is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of Impatiens. Currently, there are no available methods to detect P. obducens from asymtomatic plants, 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. In this study, we developed and standardized a FISH technique for the detection of P. obducens from Impatiens walleriana using a species-specific 24-mer oligonucleotide probe designed to targte the rRNA internal transcribed spacer 2 (ITS2). Because P. obducens cannot be propagated in vitro, we developed a custom E. coli expression vector that transcribes the P. obducens rRNA-ITS2 sequence (clone-FISH) for use as a control and to optimize hybridization conditions. The FISH assay could detect P. obducens sporiangiophores, sporangia, and oospores, and hyphae from infected I. walleriana leaves and stems. Cross-reactivity was not observed when the assay was applied to E. coli with self-ligated plasmids and non-target oomycete species. This assay may provide a valuable tool to improve monitoring of P. obducens from asymptomatic plants.