Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH)
Abstract 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 cont...
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doaj-acf430e772ef44438345ceecb8b060942020-11-24T21:37:02ZengBMCPlant Methods1746-48112018-10-011411910.1186/s13007-018-0362-zVisualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH)Catalina Salgado-Salazar0Gary R. Bauchan1Emma C. Wallace2Jo Anne Crouch3Agriculture Research Service (ARS), Mycology and Nematology Genetic Diversity and Biology Laboratory, U.S. Department of AgricultureAgriculture Research Service, Electron and Confocal Microscopy Unit, U.S. Department of AgricultureAgriculture Research Service (ARS), Mycology and Nematology Genetic Diversity and Biology Laboratory, U.S. Department of AgricultureAgriculture Research Service (ARS), Mycology and Nematology Genetic Diversity and Biology Laboratory, U.S. Department of AgricultureAbstract 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.http://link.springer.com/article/10.1186/s13007-018-0362-zOomycotaPeronosporalesOosporesDowny mildewNatural environment |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Catalina Salgado-Salazar Gary R. Bauchan Emma C. Wallace Jo Anne Crouch |
spellingShingle |
Catalina Salgado-Salazar Gary R. Bauchan Emma C. Wallace Jo Anne Crouch Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH) Plant Methods Oomycota Peronosporales Oospores Downy mildew Natural environment |
author_facet |
Catalina Salgado-Salazar Gary R. Bauchan Emma C. Wallace Jo Anne Crouch |
author_sort |
Catalina Salgado-Salazar |
title |
Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH) |
title_short |
Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH) |
title_full |
Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH) |
title_fullStr |
Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH) |
title_full_unstemmed |
Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH) |
title_sort |
visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (fish) |
publisher |
BMC |
series |
Plant Methods |
issn |
1746-4811 |
publishDate |
2018-10-01 |
description |
Abstract 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. |
topic |
Oomycota Peronosporales Oospores Downy mildew Natural environment |
url |
http://link.springer.com/article/10.1186/s13007-018-0362-z |
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