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

Salgado-Salazar, Catalina; Bauchan, Gary R.; Wallace, Emma C.; Crouch, Jo Anne

doi:10.1186/s13007-018-0362-z

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

Journal Article · 23 October 2018 · Plant Methods

DOI: https://doi.org/10.1186/s13007-018-0362-z · OSTI ID:1626917

Salgado-Salazar, Catalina ^[1]; Bauchan, Gary R. ^[2]; Wallace, Emma C. ^[3]; Crouch, Jo Anne ^[2]

US Dept. of Agriculture (USDA), Beltsville, MD (United States). Agriculture Research Service (ARS); Oak Ridge Inst. for Science and Education (ORISE), Oak Ridge, TN (United States). ARS Research Participation Program; DOE/OSTI
US Dept. of Agriculture (USDA), Beltsville, MD (United States). Agriculture Research Service (ARS)
US Dept. of Agriculture (USDA), Beltsville, MD (United States). Agriculture Research Service (ARS); Oak Ridge Inst. for Science and Education (ORISE), Oak Ridge, TN (United States). ARS Research Participation Program

Background: Plasmopara obducens is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of Impatiens that causes high crop loss. Currently, there are no available methods for the microscopic detection of P. obducens 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 P. obducens from Impatiens walleriana tissues using a species-specific 24-mer oligonucleotide probe designed to target a region of the rRNA internal transcribed spacer 2 (ITS2). Results: Since P. obducens cannot be propagated in vitro, we developed a custom E. coli expression vector that transcribes the P. obducens rRNA-ITS target sequence (clone-FISH) for use as a control and to optimize hybridization conditions. The FISH assay could detect P. obducens sporangiophores, sporangia and oospores, and hyphae from naturally infected I. walleriana leaves and stems. Cross-reactivity was not observed from plant tissue, and the assay did not react when applied to E. coli 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 P. obducens, substantially reducing the persistence and spread of this destructive plant pathogen.

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Research Organization:: Oak Ridge Associated Univ., Oak Ridge, TN (United States)

Sponsoring Organization:: Agricultural Research Service (ARS); USDA; USDOE

Grant/Contract Number:: AC05-06OR23100

OSTI ID:: 1626917

Journal Information:: Plant Methods, Journal Name: Plant Methods Journal Issue: 1 Vol. 14; ISSN 1746-4811

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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Title: Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH)

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