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Vegetable Crops Research Bulletin

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A Simple Dual Stain for Detailed Investigations of Plant-Fungal Pathogen Interactions

Marcin Nowicki1 / Małgorzata Lichocka2 / Marzena Nowakowska1 / Urszula Kłosińska1 / 1

1Research Institute of Horticulture Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland

2Institute of Biochemistry and Biophysics, Polish Academy of Sciences Pawińskiego 5a, 02-106 Warszawa, Poland

This content is open access.

Citation Information: Vegetable Crops Research Bulletin. Volume 77, Issue , Pages 61–74, ISSN (Online) 1898-7761, ISSN (Print) 1506-9427, DOI: https://doi.org/10.2478/v10032-012-0016-z, May 2013

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This article offers supplementary material which is provided at the end of the article.


Dramatic increase in confocal microscopy observation output has been gained by optimization of a simple trypan blue and aniline blue dual-stain and its application to two model pathosystems: Pseudoperonospora cubensiscucumber and Phytophthora infestans-tomato. Comparison of two dual-stain methods for confocal microscopy studies of P. cubensis-challenged cucumber leaves indicated the 'mild' approach most successful. This methodology provides simultaneous detection of different pathogen structures layered with the plant defense reactions. Moreover, ImageJ-assisted quantification of plant defense responses renders this method useful for addressing the host plant resistance reactions, as well as investigating the given isolate's pathogenicity. Application of this method for the P. infestans-challenged tomato leaf samples resulted in detection of several fungal infection structures, along with plant defense responses. The dual-stain also enabled detection of a peculiar aniline blue-sensitive material in the pathogen cell walls at the area of its hyphae emerging through the leaf stomata. Results presented herein indicate this method is applicable for detailed (possibly quantitative) investigations of multiple plant-fungal pathosystems.


Optymalizowano metodę podwójnego barwienia (błękit trypanowy i błękit anilinowy) eksplantatów zainokulowanych liści celem mikroskopowych analiz konfokalnych dwóch patosystemów: Pseudoperonospora cubensis - ogórek oraz Phytophthora infestans - pomidor. Wskutek przeprowadzonej optymalizacji, uzyskano olbrzymi wzrost wydajności analiz obydwu badanych patosystemów. Porównanie dwóch technik podwójnego barwienia, celem późniejszych analiz konfokalnych eksplantatów ogórka zainokulowanych P. cubensis, wskazuje na lepsze wyniki przy zastosowaniu „łagodnego” protokołu barwienia. Aplikacja tego protokołu pozwoliła na detekcję struktur infekcyjnych patogenów w obu badanych patosystemach, przy równoczesnych obserwacjach reakcji obronnych roślin. Ilościowe analizy obrazów reakcji odpornościowych ogórka po inokulacji P. cubensis przeprowadzone przy pomocy programu ImageJ wskazują, że metoda ta jest przydatna zarówno do charakterystyki reakcji obronnych roślin, jak i do określania poziomu patogeniczności danego izolatu. Dodatkowo, metoda ta umożliwiła detekcję specyficznego materiału o powinowactwie do błękitu anilinowego w ścianach komórkowych P. infestans, w obszarze sporulującej grzybni przerastającej aparaty szparkowe liści pomidora. Zaprezentowane wyniki wskazują na możliwość zastosowania tej metody do szczegółowych (ilościowych) analiz, również innych patosystemów

Keywords: histochemical dual-stain; cucumber downy mildew; tomato late blight; pathogen infection structures; host plant resistance; resistance reaction quantification

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